GSSG Disulfide, Glutathione OXIGLUTATIONE Oxidized Glutathione BDBM225231
BDBM233038 Dimethyl disulfide
BDBM68274 Butyl-CoA disulfide 2
Decyl-CoA disulfide 5 BDBM68278
Methyl-CoA disulfide 1 BDBM68275
Octyl-CoA disulfide 4 BDBM68277
Sec-butyl-CoA disulfide 3 BDBM68276
GLUTATHIONE BDBM50422268 GSH
BDBM50444458 BI-87F4 CHEBI:53239 BENZOTHIAZYL DISULFIDE
Diallyl disulfide 3-Allyldisulfanyl-propene BDBM50318453 CHEMBL366603
CHEMBL538665 Di(8-Phenyladenosine-5'-yl)disulfide BDBM50295367
Di(8-bromoadenosine-5'-yl)disulfide BDBM50295365 CHEMBL562056
BDBM50036005 CHEMBL422540 di(3-phenyl-1H-2-indolyl) disulfide
BDBM50295363 CHEMBL559921 (tiazofurin-5'-yl)(adenosin-5'-yl)disulfide
BDBM50295366 Di(2'-C-methyladenosine-5'-yl)disulfide CHEMBL558497
di(1,3-dimethyl-1H-2-indolyl) disulfide BDBM50036018 CHEMBL158879
BDBM50092828 S-(N-hydroxycarbamoyl)glutathione CHEMBL419969
BDBM50295364 (8-Bromoadenosine-5'-yl)(adenosine-5'-yl)disulfide CHEMBL560315
BDBM50039112 S-(N-Methyl-N-carbomyl)glutathione
S-(N-Hydroxy-N-phenylcarbamoyl)glutathione BDBM50039113
BDBM50092824 CHEMBL131578 S-(N-4chlorophenyl-N-hydroxycarbamoyl)glutathione
BDBM50092825 CHEMBL128935 S-(N-phenyl-N-hydroxycarbamoyl)glutathione
BDBM50092827 CHEMBL127840 S-(N-hexyl-N-hydroxycarbamoyl)glutathione
BDBM50092829 CHEMBL129597 S-(N-propyl-N-hydroxycarbamoyl)glutathione
BDBM50092831 CHEMBL128836 S-(N-heptyl-N-hydroxycarbamoyl)glutathione
CHEMBL128447 S-(N-butyl-N-hydroxycarbamoyl)glutathione BDBM50092822
CHEMBL129435 S-(N-methyl-N-hydroxycarbamoyl)glutathione BDBM50092823
S-(N-Methyl-N-hydroxycarbomyl)glutathione BDBM50039115 CHEMBL68824
S-(N-ethyl-N-hydroxycarbamoyl)glutathione CHEMBL128867 BDBM50092832
S-(N-pentyl-N-hydroxycarbamoyl)glutathione CHEMBL129965 BDBM50092830
BDBM50039111 S-(N-Hydroxy-N-(4-bromophenyl)carbamoyl)glutathione
S-(N-Hydroxy-N-(4-chlorophenyl)carbamoyl)glutathione BDBM50039108
N,N,N',N'-tetraethylthiuram disulfide N,N-diethyl[(diethylcarbamothioyl)disulfanyl]carbothioamide (G5) BDBM50058655 US20230414581, Compound 37 Antabuse (TN) tetraethylthioperoxydicarbonic diamide DISULFIRAM med.21724, Compound 151 1,1',1'',1'''-[disulfanediylbis(carbonothioylnitrilo)]tetraethane Disul-firam CHEMBL964 bis(diethylthiocarbamoyl) disulfide tetraethylthiuram disulfide cid_3117 1,1'-dithiobis(N,N-diethylthioformamide) US11753371, Compound II-2a-(Disulfiram) tetraethylthiuram disulphide
Bis(1-allyl-6-nitro-quinazolin-4(1H)-one-2-yl)-disulfide hydrochloride BDBM50316139 CHEMBL1091243
CHEMBL418037 disulfide-containing sulfonamide 5a BDBM11052 N-(4-sulfamoylphenyl)-2-({2-[(4-sulfamoylphenyl)carbamoyl]phenyl}disulfanyl)benzamide
disulfide-containing sulfonamide 5b N-[2-(4-sulfamoylphenyl)ethyl]-2-[(2-{[2-(4-sulfamoylphenyl)ethyl]carbamoyl}phenyl)disulfanyl]benzamide CHEMBL353498 BDBM11054
BDBM11040 disulfide-containing sulfonamide 2a N,N-(Dithiodipropionyl)bis(4-aminobenzenesulfonamide) N-(4-sulfamoylphenyl)-3-({2-[(4-sulfamoylphenyl)carbamoyl]ethyl}disulfanyl)propanamide
N,N-dimethylcarbamodithioic acid (dimethylthiocarbamoylthio) ester N,N-dimethylcarbamodithioic acid [[dimethylamino(sulfanylidene)methyl]thio] ester MLS000069752 BDBM43362 SMR000059023 cid_5455 TETRAMETHYLTHIURAM DISULFIDE dimethylcarbamothioylsulfanyl N,N-dimethylcarbamodithioate
Glutathione, N-glycylthiosuccinimide (2S)-2-amino-4-{[(1R)-2-{[1-(carboxymethyl)-2,5-dioxopyrrolidin-3-yl]sulfanyl}-1-[(carboxymethyl)carbamoyl]ethyl]carbamoyl}butanoic acid methane BDBM16441
BDBM28342 Oxidized trypanothione TSST Trypanothione disulfide (2S)-2-amino-4-{[(4R,23R)-23-[(2S)-2-amino-4-formamidobutanoic acid]-5,8,19,22-tetraoxo-1,2-dithia-6,9,13,18,21-pentaazacyclotetracosan-4-yl]carbamoyl}butanoic acid T(S)2
BDBM50295362 di(adenosin-5'-yl)disulfide CHEMBL561654 (2S,3S,4R,5R,2'S,3'S,4'R,5'R)-2,2'-[DITHIOBIS(METHYLENE)]BIS[5-(6-AMINO-9H-PURIN-9-YL)TETRAHYDROFURAN-3,4-DIOL]
N-(2-chloro-4-sulfamoylphenyl)-3-({2-[(2-chloro-4-sulfamoylphenyl)carbamoyl]ethyl}disulfanyl)propanamide disulfide-containing sulfonamide 2c N-[4-(aminosulfonyl)-2-chlorophenyl]-3-[(3-{[4-(aminosulfonyl)-2-chlorophenyl]amino}-3-oxopropyl)dithio]propanamide BDBM11044
N-[4-(aminosulfonyl)-2-fluorophenyl]-3-[(3-{[4-(aminosulfonyl)-2-fluorophenyl]amino}-3-oxopropyl)dithio]propanamide disulfide-containing sulfonamide 2b N-(2-fluoro-4-sulfamoylphenyl)-3-({2-[(2-fluoro-4-sulfamoylphenyl)carbamoyl]ethyl}disulfanyl)propanamide BDBM11042
disulfide-containing sulfonamide 2d BDBM11046 N-[2-(4-sulfamoylphenyl)ethyl]-3-[(2-{[2-(4-sulfamoylphenyl)ethyl]carbamoyl}ethyl)disulfanyl]propanamide N-{2-[4-(aminosulfonyl)phenyl]ethyl}-4-{[3-({2-[4-(aminosulfonyl)phenyl]ethyl}amino)-3-oxopropyl]dithio}butanamide
N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-3-({2-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl]ethyl}disulfanyl)propanamide N-[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]-3-[(3-{[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]amino}-3-oxopropyl)dithio]propanamide disulfide-containing sulfonamide 3 BDBM11048
S-(N-4bromophenyl-N-hydroxycarbamoyl)glutathione CHEMBL128872 BDBM50092826 (2S)-2-amino-5-{[(1R)-1-[({[(4-bromophenyl)(hydroxy)amino]carbonyl}thio)methyl]-2-(carboxyamino)-2-oxoethyl]amino}-5-oxopentanoic acid (S)-2-amino-5-((R)-3-((4-bromophenyl)hydroxycarbamoylthio)-1-(carboxymethylamino)-1-oxopropan-2-ylamino)-5-oxopentanoic acid
BDBM11050 N-[(2E)-3-methyl-5-sulfamoyl-2,3-dihydro-1,3,4-thiadiazol-2-ylidene]-3-[(2-{[(2E)-3-methyl-5-sulfamoyl-2,3-dihydro-1,3,4-thiadiazol-2-ylidene]carbamoyl}ethyl)disulfanyl]propanamide N-[(2E)-5-(aminosulfonyl)-3-methyl-1,3,4-thiadiazol-2(3H)-ylidene]-3-[(3-{[(2E)-5-(aminosulfonyl)-3-methyl-1,3,4-thiadiazol-2(3H)-ylidene]amino}-3-oxopropyl)dithio]propanamide disulfide-containing sulfonamide 4
CHEMBL411836 Ala-Gly-c(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH H-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH SRIF-14 Somatostatin-14 Ala-Gly-c-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys](SRIF-14) SOMATOSTATIN BDBM50019568 SRIF14 somatostatin(H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH) Somatostatin receptor ligand H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH (Disulfide bridge: 3-14)
- Hocart, SJ; Jain, R; Murphy, WA; Taylor, JE; Coy, DH Highly potent cyclic disulfide antagonists of somatostatin. J Med Chem 42: 1863-71 (1999)
- Ekinci, D; Cankaya, M; Gül, Ä°; Coban, TA Susceptibility of cord blood antioxidant enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase to different antibiotics: in vitro approach. J Enzyme Inhib Med Chem 28: 824-9 (2013)
- Klotz, P; Slaoui-Hasnaoui, A; Banères, JL; Duckert, JF; Rossi, JC; Kerbal, A Synthesis and glutathione S-transferase structure-affinity relationships of nonpeptide and peptidase-stable glutathione analogues. J Med Chem 41: 2278-88 (1998)
- Muttenthaler, M; Andersson, A; de Araujo, AD; Dekan, Z; Lewis, RJ; Alewood, PF Modulating oxytocin activity and plasma stability by disulfide bond engineering. J Med Chem 53: 8585-96 (2010)
- Adachi, Y; Sakimura, K; Shimizu, Y; Nakayama, M; Terao, Y; Yano, T; Asami, T Potent and selective oxytocin receptor agonists without disulfide bridges. Bioorg Med Chem Lett 27: 2331-2335 (2017)
- Kaplan, A; Stockwell, BR Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase. ACS Med Chem Lett 6: 966-971 (2015)
- Shergalis, A; Xue, D; Gharbia, FZ; Driks, H; Shrestha, B; Tanweer, A; Cromer, K; Ljungman, M; Neamati, N Characterization of Aminobenzylphenols as Protein Disulfide Isomerase Inhibitors in Glioblastoma Cell Lines. J Med Chem 63: 10263-10286 (2020)
- Purohit, MK; Scovell, I; Neschadim, A; Katsman, Y; Branch, DR; Kotra, LP Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells. Bioorg Med Chem Lett 23: 2324-7 (2013)
- Xu, P; Kaas, Q; Wu, Y; Zhu, X; Li, X; Harvey, PJ; Zhangsun, D; Craik, DJ; Luo, S Structure and Activity Studies of Disulfide-Deficient Analogues of ?O-Conotoxin GeXIVA. J Med Chem 63: 1564-1575 (2020)
- Flaumenhaft, R; Nag, PP; Pilyugina, T; Pu, J; Dandapani, S; Munoz, B; Dockendorff, C Compounds for use in methods for treating diseases or conditions mediated by protein disulfide isomerase US Patent US10064853 (2018)
- Yang, S; Shergalis, A; Lu, D; Kyani, A; Liu, Z; Ljungman, M; Neamati, N Design, Synthesis, and Biological Evaluation of Novel Allosteric Protein Disulfide Isomerase Inhibitors. J Med Chem 62: 3447-3474 (2019)
- Einsiedel, J; Schmidt, MF; Hübner, H; Gmeiner, P Development of disulfide-functionalized peptides covalently binding G protein-coupled receptors. Bioorg Med Chem 61: (2022)
- Buchholz, K; Schirmer, RH; Eubel, JK; Akoachere, MB; Dandekar, T; Becker, K; Gromer, S Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum. Antimicrob Agents Chemother 52: 183-91 (2007)
- Ge, J; Zhang, CJ; Li, L; Chong, LM; Wu, X; Hao, P; Sze, SK; Yao, SQ Small molecule probe suitable for in situ profiling and inhibition of protein disulfide isomerase. ACS Chem Biol 8: 2577-85 (2013)
- Taha, M; Ismail, NH; Imran, S; Wadood, A; Rahim, F; Al Muqarrabin, LM; Zaki, HM; Ahmat, N; Nasir, A; Khan, F Synthesis of novel disulfide and sulfone hybrid scaffolds as potent ß-glucuronidase inhibitor. Bioorg Chem 68: 15-22 (2016)
- Taha, M; Ismail, NH; Imran, S; Wadood, A; Rahim, F; Riaz, M Synthesis of potent urease inhibitors based on disulfide scaffold and their molecular docking studies. Bioorg Med Chem 23: 7211-8 (2015)
- Hoque, MA; Arai, T; Nishino, N; Kim, HJ; Ito, A; Yoshida, M Cyclic tetrapeptides with thioacetate tails or intramolecular disulfide bridge as potent inhibitors of histone deacetylases. Bioorg Med Chem Lett 22: 6770-2 (2012)
- Li, L; Liu, J; Ding, Y; Shi, Z; Peng, B; Yang, N; Hong, D; Zhang, C; Yao, C; Ge, J; Huang, W Design, synthesis and evaluation of protein disulfide isomerase inhibitors with nitric oxide releasing activity. Bioorg Med Chem Lett 30: (2020)
- Fong, J; Yuan, M; Jakobsen, TH; Mortensen, KT; Delos Santos, MM; Chua, SL; Yang, L; Tan, CH; Nielsen, TE; Givskov, M Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa. J Med Chem 60: 215-227 (2017)
- Kumprecht, L; Budesínský, M; Vondrásek, J; Vymetal, J; Cerný, J; Císarová, I; Brynda, J; Herzig, V; Koutník, P; Závada, J; Kraus, T Rigid duplex alpha-cyclodextrin reversibly connected with disulfide bonds. Synthesis and inclusion complexes. J Org Chem 74: 1082-92 (2009)
- DiMaio, J; Jaramillo, J; Wernic, D; Grenier, L; Welchner, E; Adams, J Synthesis and biological activity of atrial natriuretic factor analogues: effect of modifications to the disulfide bridge. J Med Chem 33: 661-7 (1990)
- Grieco, P; Carotenuto, A; Campiglia, P; Zampelli, E; Patacchini, R; Maggi, CA; Novellino, E; Rovero, P A new, potent urotensin II receptor peptide agonist containing a Pen residue at the disulfide bridge. J Med Chem 45: 4391-4 (2002)
- Liang, C; Cai, M; Xu, Y; Fu, W; Wu, J; Liu, Y; Liao, X; Ning, J; Li, J; Huang, M; Yuan, C Identification of Antithrombotic Natural Products Targeting the Major Substrate Binding Pocket of Protein Disulfide Isomerase. J Nat Prod 85: 1332-1339 (2022)
- Vergauwen, B; Van der Meeren, R; Dansercoer, A; Savvides, SN Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins. BMC Biochem 12: 59 (2011)
- Karaman, M; Akkemik, E; Budak, H; Ciftci, M In vitro effects of some drugs on human erythrocyte glutathione reductase. J Enzyme Inhib Med Chem 27: 18-23 (2012)
- Lyttle, MH; Hocker, MD; Hui, HC; Caldwell, CG; Aaron, DT; Engqvist-Goldstein, A; Flatgaard, JE; Bauer, KE Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis. J Med Chem 37: 189-94 (1994)
- Misicka, A; Lipkowski, AW; Horvath, R; Davis, P; Yamamura, HI; Porreca, F; Hruby, VJ Design of cyclic deltorphins and dermenkephalins with a disulfide bridge leads to analogues with high selectivity for delta-opioid receptors. J Med Chem 37: 141-5 (1994)
- Tsuchida, K; Chaki, H; Takakura, T; Yokotani, J; Aikawa, Y; Shiozawa, S; Gouda, H; Hirono, S Design, synthesis, and biological evaluation of new cyclic disulfide decapeptides that inhibit the binding of AP-1 to DNA. J Med Chem 47: 4239-46 (2004)
- Xu, YX; Huang, YY; Song, RR; Ren, YL; Chen, X; Zhang, C; Mao, F; Li, XK; Zhu, J; Ni, SS; Wan, J; Li, J Development of disulfide-derived fructose-1,6-bisphosphatase (FBPase) covalent inhibitors for the treatment of type 2 diabetes. Eur J Med Chem 203: (2020)
- Andersson, H; Demaegdt, H; Vauquelin, G; Lindeberg, G; Karlén, A; Hallberg, M; Erdélyi, M; Hallberg, A Disulfide cyclized tripeptide analogues of angiotensin IV as potent and selective inhibitors of insulin-regulated aminopeptidase (IRAP). J Med Chem 53: 8059-71 (2010)
- Dong, M; Pinon, DI; Bordner, AJ; Miller, LJ Elucidation of the active conformation of the amino terminus of receptor-bound secretin using intramolecular disulfide bond constraints. Bioorg Med Chem Lett 20: 6040-4 (2010)
- Rais, R; Hoover, R; Wozniak, K; Rudek, MA; Tsukamoto, T; Alt, J; Rojas, C; Slusher, BS Reversible disulfide formation of the glutamate carboxypeptidase II inhibitor E2072 results in prolonged systemic exposures in vivo. Drug Metab Dispos 40: 2315-23 (2012)
- Petrelli, R; Sham, YY; Chen, L; Felczak, K; Bennett, E; Wilson, D; Aldrich, C; Yu, JS; Cappellacci, L; Franchetti, P; Grifantini, M; Mazzola, F; Di Stefano, M; Magni, G; Pankiewicz, KW Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues. Bioorg Med Chem 17: 5656-64 (2009)
- Chen, L; Zoulíková, I; Slaninová, J; Barany, G Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: directed methods for the construction of disulfide and trisulfide bridges in peptides. J Med Chem 40: 864-76 (1997)
- Chen, J; Jiang, CS; Ma, WQ; Gao, LX; Gong, JX; Li, JY; Li, J; Guo, YW The first synthesis of natural disulfide bruguiesulfurol and biological evaluation of its derivatives as a novel scaffold for PTP1B inhibitors. Bioorg Med Chem Lett 23: 5061-5 (2013)
- Brophy, PM; Campbell, AM; van Eldik, AJ; Teesdale-Spittle, PH; Liebau, E; Wang, MF Beta-carbonyl substituted glutathione conjugates as inhibitors Of O. volvulus GST2. Bioorg Med Chem Lett 10: 979-81 (2000)
- Xie, Y; Tummala, P; Oakley, AJ; Deora, GS; Nakano, Y; Rooke, M; Cuellar, ME; Strasser, JM; Dahlin, JL; Walters, MA; Casarotto, MG; Board, PG; Baell, JB Development of Benzenesulfonamide Derivatives as Potent Glutathione Transferase Omega-1 Inhibitors. J Med Chem 63: 2894-2914 (2020)
- Qian, YM; Song, WC; Cui, H; Cole, SP; Deeley, RG Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1. J Biol Chem 276: 6404-11 (2001)
- Larson, DL; Hua, M; Takemori, AE; Portoghese, PS Possible contribution of a glutathione conjugate to the long-duration action of beta-funaltrexamine. J Med Chem 36: 3669-73 (1994)
- De Simone, G; Vitale, RM; Di Fiore, A; Pedone, C; Scozzafava, A; Montero, JL; Winum, JY; Supuran, CT Carbonic anhydrase inhibitors: Hypoxia-activatable sulfonamides incorporating disulfide bonds that target the tumor-associated isoform IX. J Med Chem 49: 5544-51 (2006)
- Fridkin, G; Maina, T; Nock, BA; Blat, D; Lev-Goldman, V; Scolnik, Y; Kapitkovski, A; Vachutinsky, Y; Shechter, Y; Levy, Y Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs. Bioorg Med Chem 19: 798-806 (2011)
- Poras, H; Bonnard, E; Fournié-Zaluski, MC; Roques, BP Modulation of disulfide dual ENKephalinase inhibitors (DENKIs) activity by a transient N-protection for pain alleviation by oral route. Eur J Med Chem 102: 58-67 (2015)
- Davioud-Charvet, E; Delarue, S; Biot, C; Schwöbel, B; Boehme, CC; Müssigbrodt, A; Maes, L; Sergheraert, C; Grellier, P; Schirmer, RH; Becker, K A prodrug form of a Plasmodium falciparum glutathione reductase inhibitor conjugated with a 4-anilinoquinoline. J Med Chem 44: 4268-76 (2001)
- Renes, J; de Vries, EG; Nienhuis, EF; Jansen, PL; Müller, M ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1. Br J Pharmacol 126: 681-8 (1999)
- Maeda, DY; Mahajan, SS; Atkins, WM; Zebala, JA Bivalent inhibitors of glutathione S-transferase: the effect of spacer length on isozyme selectivity. Bioorg Med Chem Lett 16: 3780-3 (2006)
- Sentürk, M; Talaz, O; Ekinci, D; Cavdar, H; Küfrevioglu, OI In vitro inhibition of human erythrocyte glutathione reductase by some new organic nitrates. Bioorg Med Chem Lett 19: 3661-3 (2009)
- Schönleben-Janas, A; Kirsch, P; Mittl, PR; Schirmer, RH; Krauth-Siegel, RL Inhibition of human glutathione reductase by 10-arylisoalloxazines: crystalline, kinetic, and electrochemical studies. J Med Chem 39: 1549-54 (1996)
- Bai, J; Lai, L; Yeo, HC; Goh, BC; Tan, TM Multidrug resistance protein 4 (MRP4/ABCC4) mediates efflux of bimane-glutathione. Int J Biochem Cell Biol 36: 247-57 (2004)
- Zhang, R; Xiao, N; Xu, Q; Gong, Q; Kong, F; Jiang, H Pleiotropic effects of a mitochondrion-targeted glutathione reductase inhibitor on restraining tumor cells. Eur J Med Chem 248: (2023)
- Kamiya, D; Uchihata, Y; Ichikawa, E; Kato, K; Umezawa, K Reversal of anticancer drug resistance by COTC based on intracellular glutathione and glyoxalase I. Bioorg Med Chem Lett 15: 1111-4 (2005)
- Hoque, MA; Islam, MN; Islam, MS; Kato, T; Nishino, N; Ito, A; Yoshida, M Design and synthesis of CHAP31, trapoxin B and HC-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors. Bioorg Med Chem 22: 3850-5 (2014)
- Liu, Y; Xie, Z; Zhao, D; Zhu, J; Mao, F; Tang, S; Xu, H; Luo, C; Geng, M; Huang, M; Li, J Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J Med Chem 60: 2227-2244 (2017)
- Çakmak, R; Durdagi, S; Ekinci, D; Sentürk, M; Topal, G Design, synthesis and biological evaluation of novel nitroaromatic compounds as potent glutathione reductase inhibitors. Bioorg Med Chem Lett 21: 5398-402 (2011)
- Mangoyi, R; Hayeshi, R; Ngadjui, B; Ngandeu, F; Bezabih, M; Abegaz, B; Razafimahefa, S; Rasoanaivo, P; Mukanganyama, S Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products. J Enzyme Inhib Med Chem 25: 854-62 (2010)
- Amssoms, K; Oza, SL; Augustyns, K; Yamani, A; Lambeir, AM; Bal, G; Van der Veken, P; Fairlamb, AH; Haemers, A Glutathione-like tripeptides as inhibitors of glutathionylspermidine synthetase. Part 2: substitution of the glycine part. Bioorg Med Chem Lett 12: 2703-5 (2002)
- Kobzar, O; Shulha, Y; Buldenko, V; Cherenok, S; Silenko, O; Kalchenko, V; Vovk, A Inhibition of glutathione S-transferases by photoactive calix[4]arene ?-ketophosphonic acids. Bioorg Med Chem Lett 77: (2022)
- Muleya, V; Hayeshi, R; Ranson, H; Abegaz, B; Bezabih, MT; Robert, M; Ngadjui, BT; Ngandeu, F; Mukanganyama, S Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro. J Enzyme Inhib Med Chem 23: 391-9 (2008)
- Song, GY; Zheng, XG; Kim, Y; You, YJ; Sok, DE; Ahn, BZ Naphthazarin derivatives (II): formation of glutathione conjugate, inhibition of DNA topoisomerase-I and cytotoxicity. Bioorg Med Chem Lett 9: 2407-12 (1999)
- Xie, Y; Dahlin, JL; Oakley, AJ; Casarotto, MG; Board, PG; Baell, JB Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example. J Med Chem 61: 7448-7470 (2018)
- Axarli, I; Labrou, NE; Petrou, C; Rassias, N; Cordopatis, P; Clonis, YD Sulphonamide-based bombesin prodrug analogues for glutathione transferase, useful in targeted cancer chemotherapy. Eur J Med Chem 44: 2009-16 (2009)
- Jedlitschky, G; Leier, I; Buchholz, U; Barnouin, K; Kurz, G; Keppler, D Transport of glutathione, glucuronate, and sulfate conjugates by the MRP gene-encoded conjugate export pump. Cancer Res 56: 988-94 (1996)
- García-Aranda, MI; Mirassou, Y; Gautier, B; Martín-Martínez, M; Inguimbert, N; Vidal, M; García-López, MT; Jiménez, MA; González-Muñiz, R; Pérez de Vega, MJ Disulfide and amide-bridged cyclic peptide analogues of the VEGF8¿?¿9¿? fragment: synthesis, conformational analysis and biological evaluation. Bioorg Med Chem 19: 7526-33 (2011)
- Ye, Y; Xu, B; Nikiforovich, GV; Bloch, S; Achilefu, S Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging. Bioorg Med Chem Lett 21: 2116-20 (2011)
- Ambo, A; Hamazaki, N; Yamada, Y; Nakata, E; Sasaki, Y Structure-activity studies on nociceptin analogues: ORL1 receptor binding and biological activity of cyclic disulfide-containing analogues of nociceptin peptides. J Med Chem 44: 4015-8 (2001)
- Leier, I; Jedlitschky, G; Buchholz, U; Center, M; Cole, SP; Deeley, RG; Keppler, D ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump. Biochem J 314: 433-7 (1996)
- Rotili, D; De Luca, A; Tarantino, D; Pezzola, S; Forgione, M; Morozzo Della Rocca, B; Falconi, M; Mai, A; Caccuri, AM Synthesis and structure--activity relationship of new cytotoxic agents targeting human glutathione-S-transferases. Eur J Med Chem 89: 156-71 (2014)
- Lherbet, C; Gravel, C; Keillor, JW Synthesis of S-alkyl L-homocysteine analogues of glutathione and their kinetic studies with gamma-glutamyl transpeptidase. Bioorg Med Chem Lett 14: 3451-5 (2004)
- de Lucio, H; Revuelto, A; Carriles, AA; de Castro, S; García-González, S; García-Soriano, JC; Alcón-Calderón, M; Sánchez-Murcia, PA; Hermoso, JA; Gago, F; Camarasa, MJ; Jiménez-Ruiz, A; Velázquez, S Identification of 1,2,3-triazolium salt-based inhibitors of Leishmania infantum trypanothione disulfide reductase with enhanced antileishmanial potency in cellulo and increased selectivity. Eur J Med Chem 244: (2022)
- Manral, A; Saini, V; Meena, P; Tiwari, M Multifunctional novel Diallyl disulfide (DADS) derivatives withß-amyloid-reducing, cholinergic, antioxidant and metal chelating properties for the treatment of Alzheimer's disease. Bioorg Med Chem 23: 6389-403 (2015)
- Biot, C; Bauer, H; Schirmer, RH; Davioud-Charvet, E 5-substituted tetrazoles as bioisosteres of carboxylic acids. Bioisosterism and mechanistic studies on glutathione reductase inhibitors as antimalarials. J Med Chem 47: 5972-83 (2004)
- PubChem, PC Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in the presence of Glutathione. PubChem Bioassay (2007)
- McNaughton, M; Engman, L; Birmingham, A; Powis, G; Cotgreave, IA Cyclodextrin-derived diorganyl tellurides as glutathione peroxidase mimics and inhibitors of thioredoxin reductase and cancer cell growth. J Med Chem 47: 233-9 (2004)
- Wang, H; Wang, C; Li, B; Zheng, C; Liu, G; Liu, Z; Zhang, L; Xu, P Discovery of ML210-Based glutathione peroxidase 4 (GPX4) degrader inducing ferroptosis of human cancer cells. Eur J Med Chem 254: (2023)
- Amssoms, K; Oza, SL; Ravaschino, E; Yamani, A; Lambeir, A; Rajan, P; Bal, G; Rodriguez, J; Fairlamb, AH; Augustyns, K; Haemers, A Glutathione-like tripeptides as inhibitors of glutathionylspermidine synthetase. Part 1: Substitution of the glycine carboxylic acid group. Bioorg Med Chem Lett 12: 2553-6 (2002)
- Procopio, A; Alcaro, S; Cundari, S; De Nino, A; Ortuso, F; Sacchetta, P; Pennelli, A; Sindona, G Molecular modeling, synthesis, and preliminary biological evaluation of glutathione-S-transferase inhibitors as potential therapeutic agents. J Med Chem 48: 6084-9 (2005)
- Juang, YP; Tsai, JY; Gu, WL; Hsu, HC; Lin, CL; Wu, CC; Liang, PH Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition. J Med Chem 67: 3626-3642 (2024)
- Zhang, M; Xu, B; Li, N; Liu, H; Shi, X; Zhang, Q; Shi, Y; Xu, K; Xiao, J; Chen, D; Zhu, H; Sun, Y; Zhang, T; Zhang, R; Fang, Q Synthesis and Biological Characterization of Cyclic Disulfide-Containing Peptide Analogs of the Multifunctional Opioid/Neuropeptide FF Receptor Agonists That Produce Long-Lasting and Nontolerant Antinociception. J Med Chem 63: 15709-15725 (2020)
- Schulz, R; Emmrich, T; Lemmerhirt, H; Leffler, U; Sydow, K; Hirt, C; Kiefer, T; Link, A; Bednarski, PJ Identification of a glutathione peroxidase inhibitor that reverses resistance to anticancer drugs in human B-cell lymphoma cell lines. Bioorg Med Chem Lett 22: 6712-5 (2012)
- Morrow, CS; Peklak-Scott, C; Bishwokarma, B; Kute, TE; Smitherman, PK; Townsend, AJ Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux. Mol Pharmacol 69: 1499-505 (2006)
- Joseph, E; Ganem, B; Eiseman, JL; Creighton, DJ Selective inhibition of MCF-7(piGST) breast tumors using glutathione transferase-derived 2-methylene-cycloalkenones. J Med Chem 48: 6549-52 (2005)
- Koutsoumpli, GE; Dimaki, VD; Thireou, TN; Eliopoulos, EE; Labrou, NE; Varvounis, GI; Clonis, YD Synthesis and study of 2-(pyrrolesulfonylmethyl)-N-arylimines: a new class of inhibitors for human glutathione transferase A1-1. J Med Chem 55: 6802-13 (2012)
- Perperopoulou, FD; Tsoungas, PG; Thireou, TN; Rinotas, VE; Douni, EK; Eliopoulos, EE; Labrou, NE; Clonis, YD 2,2'-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1. Bioorg Med Chem 22: 3957-70 (2014)
- Wu, Z; Minhas, GS; Wen, D; Jiang, H; Chen, K; Zimniak, P; Zheng, J Design, synthesis, and structure-activity relationships of haloenol lactones: site-directed and isozyme-selective glutathione S-transferase inhibitors. J Med Chem 47: 3282-94 (2004)
- Wang, CH; Wu, HT; Cheng, HM; Yen, TJ; Lu, IH; Chang, HC; Jao, SC; Shing, TK; Li, WS Inhibition of glutathione S-transferase M1 by new gabosine analogues is essential for overcoming cisplatin resistance in lung cancer cells. J Med Chem 54: 8574-81 (2011)
- Hu, J; An, B; Pan, T; Li, Z; Huang, L; Li, X Design, synthesis, and biological evaluation of histone deacetylase inhibitors possessing glutathione peroxidase-like and antioxidant activities against Alzheimer's disease. Bioorg Med Chem 26: 5718-5729 (2018)
- Priebe, W; Krawczyk, M; Kuo, MT; Yamane, Y; Savaraj, N; Ishikawa, T Doxorubicin- and daunorubicin-glutathione conjugates, but not unconjugated drugs, competitively inhibit leukotriene C4 transport mediated by MRP/GS-X pump. Biochem Biophys Res Commun 247: 859-63 (1998)
- Murthy, NS; Bakeris, T; Kavarana, MJ; Hamilton, DS; Lan, Y; Creighton, DJ S-(N-aryl-N-hydroxycarbamoyl)glutathione derivatives are tight-binding inhibitors of glyoxalase I and slow substrates for glyoxalase II. J Med Chem 37: 2161-6 (1994)
- Luo, Z; Liang, L; Sheng, J; Pang, Y; Li, J; Huang, L; Li, X Synthesis and biological evaluation of a new series of ebselen derivatives as glutathione peroxidase (GPx) mimics and cholinesterase inhibitors against Alzheimer's disease. Bioorg Med Chem 22: 1355-61 (2014)
- Kaltner, H; Szabó, T; Fehér, K; André, S; Balla, S; Manning, JC; Szilágyi, L; Gabius, HJ Bivalent O-glycoside mimetics with S/disulfide/Se substitutions and aromatic core: Synthesis, molecular modeling and inhibitory activity on biomedically relevant lectins in assays of increasing physiological relevance. Bioorg Med Chem 25: 3158-3170 (2017)
- Liu, Q; Liu, Z; Hua, W; Gou, S Discovery of 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol Derivatives as Glutathione Transferase Inhibitors with Favorable Selectivity and Tolerated Toxicity. J Med Chem 64: 1701-1712 (2021)
- Chen, T; Leng, J; Tan, J; Zhao, Y; Xie, S; Zhao, S; Yan, X; Zhu, L; Luo, J; Kong, L; Yin, Y Discovery of Novel Potent Covalent Glutathione Peroxidase 4 Inhibitors as Highly Selective Ferroptosis Inducers for the Treatment of Triple-Negative Breast Cancer. J Med Chem 66: 10036-10059 (2023)
- Dai, W; Samanta, S; Xue, D; Petrunak, EM; Stuckey, JA; Han, Y; Sun, D; Wu, Y; Neamati, N Structure-Based Design of N-(5-Phenylthiazol-2-yl)acrylamides as Novel and Potent Glutathione S-Transferase Omega 1 Inhibitors. J Med Chem 62: 3068-3087 (2019)
- Wen, J; Bao, Y; Niu, Q; Yang, J; Fan, Y; Li, J; Jing, Y; Zhao, L; Liu, D Identification of N-(6-mercaptohexyl)-3-(4-pyridyl)-1H-pyrazole-5-carboxamide and its disulfide prodrug as potent histone deacetylase inhibitors with in vitro and in vivo anti-tumor efficacy. Eur J Med Chem 109: 350-9 (2016)
- Seefeldt, T; Dwivedi, C; Peitz, G; Herman, J; Carlson, L; Zhang, Z; Guan, X 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)- phenylcarbamoylsulfanyl]propionic acid and its derivatives as a novel class of glutathione reductase inhibitors. J Med Chem 48: 5224-31 (2005)
- Bülbül, M; Erat, M Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes. J Enzyme Inhib Med Chem 23: 418-23 (2008)
- Maciag, AE; Holland, RJ; Kim, Y; Kumari, V; Luthers, CE; Sehareen, WS; Biswas, D; Morris, NL; Ji, X; Anderson, LM; Saavedra, JE; Keefer, LK Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells. J Med Chem 57: 2292-302 (2014)
- Yang, X; Liu, G; Li, H; Zhang, Y; Song, D; Li, C; Wang, R; Liu, B; Liang, W; Jing, Y; Zhao, G Novel oxadiazole analogues derived from ethacrynic acid: design, synthesis, and structure-activity relationships in inhibiting the activity of glutathione S-transferase P1-1 and cancer cell proliferation. J Med Chem 53: 1015-22 (2010)
- Liu, SJ; Zhao, Q; Peng, C; Mao, Q; Wu, F; Zhang, FH; Feng, QS; He, G; Han, B Design, synthesis, and biological evaluation of nitroisoxazole-containing spiro[pyrrolidin-oxindole] derivatives as novel glutathione peroxidase 4/mouse double minute 2 dual inhibitors that inhibit breast adenocarcinoma cell proliferation. Eur J Med Chem 217: (2021)
- Niinuma, K; Takenaka, O; Horie, T; Kobayashi, K; Kato, Y; Suzuki, H; Sugiyama, Y Kinetic analysis of the primary active transport of conjugated metabolites across the bile canalicular membrane: comparative study of S-(2,4-dinitrophenyl)-glutathione and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl)benzothiazole glucuronide. J Pharmacol Exp Ther 282: 866-72 (1997)
- ChEMBL_303536 (CHEMBL877454) Inhibitory concentration against human glutathione reductase (In presence of glutathione disulfide)
- ChEMBL_2302231 Inhibition of human glutathione reductase
- ChEMBL_72608 (CHEMBL681067) Inhibition of Glutathione reductase
- ChEMBL_72609 (CHEMBL681068) Inhibition of glutathione reductase
- ChEMBL_1284129 (CHEMBL3107513) Inhibition of human recombinant glutathione reductase using glutathione as substrate preincubated for 30 mins by colorimetric assay
- ChEMBL_2340856 Inhibition of GPX4 (unknown origin) incubated for 1 hrs in presence of glutathione and glutathione reductase by microplate reader analysis
- ChEMBL_2315822 Inhibition of glutathione reductase (unknown origin)
- ChEMBL_360715 (CHEMBL869332) Inhibition of human glutathione reductase
- ChEMBL_453943 (CHEMBL885947) Inhibition of human glutathione reductase
- ChEMBL_770791 (CHEMBL1837596) Inhibition of human glutathione reductase
- ChEBML_72596 The compound was evaluated for inhibition of human Glutathione reductase
- ChEMBL_571638 (CHEMBL1031293) Inhibition of human erythrocyte glutathione reductase
- ChEMBL_700053 (CHEMBL1646830) Inhibition of yeast glutathione reductase by spectrophotometry
- ChEMBL_72474 (CHEMBL685507) In vitro inhibition of human Glutathione Reductase
- ChEMBL_729212 (CHEMBL1694676) Inhibition of human glutathione reductase by spectrophotometer
- ChEMBL_767322 (CHEMBL1825434) Inhibition of human erythrocyte Glutathione reductase
- ChEMBL_1463623 (CHEMBL3398941) Inhibition of Trypanosoma cruzi trypanothione reductase assessed as reduction of trypanothione disulfide by spectrophotometrically
- ChEBML_72477 Inhibitory concentration against human Glutathione reductase was determined
- ChEMBL_154682 (CHEMBL767201) In vitro inhibition of Plasmodium falciparum Glutathione Reductase
- ChEMBL_2195310 (CHEMBL5107670) Inhibition of rat glutathione reductase by spectrophotometric method
- ChEMBL_302540 (CHEMBL827391) Inhibitory constant against human glutathione reductase
- ChEMBL_302633 (CHEMBL839924) Inhibitory concentration against human glutathione reductase
- ChEMBL_305429 (CHEMBL830922) Inhibitory concentration against human glutathione reductase
- ChEMBL_305886 (CHEMBL832901) Inhibitory concentration against human glutathione reductase
- ChEMBL_321315 (CHEMBL881411) Inhibitory concentration against human glutathione reductase
- ChEMBL_729209 (CHEMBL1694673) Inhibition of Plasmodium falciparum glutathione S-transferase
- ChEMBL_729210 (CHEMBL1694674) Inhibition of human glutathione S-transferase P
- ChEMBL_2340880 Covalent inhibition of GPX4 (unknown origin) assessed as inhibition constant incubated for 30 to 90 mins in presence of glutathione, glutathione reductase and NADPH by microplate reader analysis
- ChEBML_72450 Inhibitory activity against human Glutathione S-transferase P
- ChEMBL_302908 (CHEMBL876366) Inhibitory concentration against Plasmodium falciparum glutathione reductase
- ChEMBL_306283 (CHEMBL828426) Inhibitory concentration against Plasmodium falciparum glutathione reductase
- ChEMBL_496923 (CHEMBL1002018) Inhibition of CDK4 by radioactive glutathione plate-binding assay
- ChEMBL_574903 (CHEMBL1023600) Inhibition of human recombinant glutathione reductase at pH 6.9
- ChEMBL_303331 (CHEMBL877448) Inhibitory concentration against human glutathione reductase (In presence of NADPH)
- ChEMBL_72308 (CHEMBL686166) Affinity towards recombinant Glutathione S-transferase (GST) Enzyme.
- ChEMBL_72453 (CHEMBL686008) Inhibitory activity for human Glutathione S-transferase P was determined
- ChEMBL_72595 (CHEMBL681183) Inhibitory activity against Human Erythrocyte glutathione reductase (GR).
- ChEMBL_878322 (CHEMBL2185950) Noncompetitive inhibition of Trypanosoma brucei TR using varying levels of trypanothione disulfide by Lineweaver-Burk plot
- ChEMBL_923296 (CHEMBL3077129) Inhibition of Homo sapiens (human) MRP1 expressed in Sf9 cell membranes assessed as decrease in N-ethyl-maleimide-glutathione-induced inorganic phosphate production by spectrophotometric analysis in presence of glutathione
- ChEMBL_431014 (CHEMBL916869) Inhibition of human glutathione reductase in presence of 500 uM GSSG substrate
- ChEMBL_431015 (CHEMBL916870) Inhibition of human glutathione reductase in presence of 50 uM GSSG substrate
- ChEMBL_72328 (CHEMBL685228) Inhibition of p53 binding to Glutathione S-transferase 2 (hdm2-GST)
- ChEMBL_72451 (CHEMBL686006) Inhibitory activity was measured on recombinant human Glutathione S-transferase P
- ChEMBL_72454 (CHEMBL686009) Inhibitory activity for human glutathione S-transferase-pi isozyme was determined
- ChEMBL_861052 (CHEMBL2166452) Inhibition of GPx1 using gultathione as substrate by glutathione reductase-coupled assay
- ChEMBL_1365745 (CHEMBL3296171) Inhibition of rat recombinant glutathione reductase after 30 mins by DTNB assay
- ChEMBL_72446 (CHEMBL682616) Inhibitory activity was measured on recombinant human Glutathione-S-transferase A1 enzyme
- ChEMBL_72447 (CHEMBL682617) Inhibitory activity was measured on recombinant human Glutathione-S-transferase M1a enzyme
- ChEMBL_72448 (CHEMBL682618) Inhibitory activity was measured on recombinant human Glutathione S-transferase Mu 1
- ChEMBL_72449 (CHEMBL682619) Inhibitory activity was measured on recombinant human Glutathione S-transferase Mu 2
- ChEMBL_72475 (CHEMBL685508) Inhibitory activity against human glutathione reductase in presence of 100 uM GSSG
- ChEMBL_72613 (CHEMBL681072) In vitro inhibitory activity against glutathione reductase (GR) from bakers yeast
- ChEMBL_210045 (CHEMBL812446) Inhibitory activity against Trypanosoma cruzi trypanothione disulfide reductase, assay in presence of 57 uM T(S)2.
- ChEBML_72327 The compound was tested for it's inhibitory activity against Onchocerca volvulus Glutathione S-transferase 2
- ChEMBL_2103008 (CHEMBL4811511) Inhibition of Schistosoma mansoni thioredoxin glutathione reductase incubated for 30 mins by DTNB assay
- ChEMBL_306528 (CHEMBL827279) Inhibitory concentration against human glutathione reductase in the presence of 200 uM exogenous NADP
- ChEMBL_1705921 (CHEMBL4057154) Competitive inhibition of Trypanosoma cruzi trypanothione reductase using varying levels of trypanothione disulfide as substrate by Lineweaver--burk plot analysis
- ChEMBL_305846 (CHEMBL829587) Inhibition of yeast glutathione reductase over 30 minutes pH 7.4 at 25 degree C
- ChEMBL_306715 (CHEMBL832300) Inhibitory concentration against human glutathione reductase in the absence of glucose-6-phosphate dehydrogenase (G6PDH)
- ChEMBL_579784 (CHEMBL1059685) Inhibition of human GSTA1-1 expressed in Escherichia coli BL21 (DE3) by glutathione competitive assay
- ChEMBL_1978319 (CHEMBL4611454) Activation of Nrf2 in human spinal cord astrocytes assessed as increase in glutathione level after 20 hrs
- ChEMBL_303237 (CHEMBL827202) Inhibition of yeast glutathione reductase for 30 minutes pH 7.4 at 25 degree C at 0.2 mM
- ChEMBL_767323 (CHEMBL1825435) Competitive inhibition of human erythrocyte Glutathione reductase using GSSG substrate by Lineweaver-Burk plot analysis
- ChEMBL_1720207 (CHEMBL4135207) Inhibition of Trypanosoma cruzi trypanothione reductase assessed as reduction in NADPH consumption using varying levels of trypanothione disulfide as substrate by Lineweaver-Burk plot analysis
- ChEBML_70497 Compound was tested for its ability to inhibit binding of [3H]- Glc1Man9 GlcNAc2 to GST-fused calnexin immobilized on glutathione-agarose
- ChEMBL_1355172 (CHEMBL3280975) Competitive inhibition of glyoxalase-1 (unknown origin) using methylglyoxal and glutathione as substrate by Lineweaver-Burk plot analysis
- ChEMBL_767324 (CHEMBL1825436) Non-Competitive inhibition of human erythrocyte Glutathione reductase using GSSG substrate by Lineweaver-Burk plot analysis
- ChEMBL_878321 (CHEMBL2185949) Mixed type inhibition of Trypanosoma brucei TR using varying levels of trypanothione disulfide assessed as inhibition constant for enzyme-inhibitor complex by Lineweaver-Burk plot
- ChEMBL_2103004 (CHEMBL4811507) Inhibition of Schistosoma mansoni thioredoxin glutathione reductase incubated for 50 mins by thio-glo reagent based fluorescence method
- ChEMBL_306096 (CHEMBL830871) Inhibition of yeast glutathione reductase done for 30 minutes in pH 7.4 at 25 degree C with compound (0.2 mM)
- ChEMBL_326139 (CHEMBL864515) Inhibition of poly(Glu4-Tyr)peptide phosphorylation by recombinant VEGFR2 at 10 uM ATP and in presence of 100 uM glutathione
- ChEMBL_431016 (CHEMBL916871) Inhibition of human glutathione reductase after 5 mins pre-incubation with NADPH in presence of 500 uM GSSG substrate
- ChEMBL_593692 (CHEMBL1044145) Inhibition of human H-PGDS expressed in Escherichia coli BL21 assessed as rate of glutathione-chloro-dinitro benzene conjugation
- ChEMBL_873195 (CHEMBL2183783) Inhibition of ATM isolated from human HeLa cell extract using glutathione S-transferase-p53N66 as substrate by ELISA
- ChEMBL_1720205 (CHEMBL4135205) Non-competitive inhibition of Trypanosoma cruzi trypanothione reductase assessed as reduction in NADPH consumption using varying levels of trypanothione disulfide as substrate by Lineweaver-Burk plot analysis
- ChEMBL_1720206 (CHEMBL4135206) Mixed-type inhibition of Trypanosoma cruzi trypanothione reductase assessed as reduction in NADPH consumption using varying levels of trypanothione disulfide as substrate by Lineweaver-Burk plot analysis
- ChEMBL_878320 (CHEMBL2185948) Mixed type inhibition of Trypanosoma brucei TR using varying levels of trypanothione disulfide assessed as inhibition constant for enzyme-substrate-inhibitor complex by Lineweaver-Burk plot
- ChEMBL_1455608 (CHEMBL3365309) Inhibition of Schistosoma mansoni recombinant thioredoxin glutathione reductase assessed as 5-thio-2nitrobezoic acid formation after 3 mins
- ChEMBL_321469 (CHEMBL880410) Inhibition against glutathione S-transferase pi using GSH (0.1-3mM), 1 mM 1-chloro-2,4-dinitrobenzene
- ChEMBL_507857 (CHEMBL951703) Inhibition of disulfide bond cross-linking formation in rat M3'(3C)-Xa receptor A91C/T549C mutant expressed in african green monkey COS7 cells by scanning densitometry
- ChEMBL_507858 (CHEMBL951704) Inhibition of disulfide bond cross-linking formation in rat M3'(3C)-Xa receptor F92C/F550C mutant expressed in african green monkey COS7 cells by scanning densitometry
- ChEMBL_507861 (CHEMBL951707) Stimulation of disulfide bond cross-linking formation in rat M3'(3C)-Xa receptor A91C/T549C mutant expressed in african green monkey COS7 cells by scanning densitometry
- ChEMBL_507862 (CHEMBL951708) Stimulation of disulfide bond cross-linking formation in rat M3'(3C)-Xa receptor F92C/F550C mutant expressed in african green monkey COS7 cells by scanning densitometry
- ChEMBL_2127684 (CHEMBL4837029) Inhibition of Escherichia coli DsbA using DOTA/Eu(III)-CQQGFDGTQNSCK-MCA as substrate in presence of oxidized glutathione measured by fluorescence analysis
- ChEMBL_221516 (CHEMBL841499) Binding affinity against p56 Lck tyrosine kinase SH2 domain was measured using glutathione S-transferase (GST) fusion protein
- ChEMBL_320920 (CHEMBL881347) Inhibition constant against glutathione S-transferase pi using GSH (0.1-3mM), 1 mM 1-chloro-2,4-dinitrobenzene
- ChEMBL_1878644 (CHEMBL4380038) Inhibition of rat lens ALR2 using 4-hydroxynonenal glutathione as substrate incubated for 1 min and measured up to 4 mins by spectrophotometric analysis
- ChEMBL_2108109 (CHEMBL4816784) Inhibition of Glyoxalase-1 (unknown origin) assessed as inhibition of S-D-lactoylglutathione formation using MG and reduced glutathione as substrate by spectrophotometric method
- ChEMBL_885082 (CHEMBL2212266) Inhibition of human recombinant GLO1 expressed in Escherichia coli BL21 assessed as decrease in reduced glutathione level after 1 hr by Ellman's method
- ChEMBL_2026867 (CHEMBL4681025) Agonist activity at glutathione transferase-tagged human FXR-LBD using biotinylated Src-1 peptide incubated for 30 mins by recruitment coactivator assay
- ChEMBL_2315524 Agonist activity at glutathione transferase-tagged human FXR ligand binding domain assessed as biotinylated Src1 peptide recruitment incubated for 30 mins by AlphaScreen assay
- ChEMBL_801918 (CHEMBL1947584) Non-competitive inhibition of recombinant Leishmania infantum MHOM/MA67ITMAP263 trypanothione reductase assessed as inhibition of NADPH consumption using trypanothione disulfide as substrate at 1 uM by Michaelis-Menten and Lineweaver-Burk plot analysis
- Glutathione Peroxidase Activity Assay Glutathione peroxidase was assayed in a l-ml system containing 0.1 M potassium phosphate buffer, pH 7.0, 0.2 mM NADPH, 1 i.u. glutathione reductase, 4 mM GSH,4 mM EDTA, 4 mM sodium azide. The reaction mixture was incubated at 37°C for 10 min after which 10 μl of 10 mM t-butyl hydroperoxide were added to start the reaction. No t-butyl hydroperoxide was added to the blank cuvette. The rate of reaction was measured at 37°C by following the decrease in the absorbance at 340 nm using a spectrophotometer.
- ChEMBL_1477164 (CHEMBL3428604) Inhibition human HPGDS expressed in Escherichia coli assessed as reduction in GST enzymatic activity using MCBL and glutathione incubated for 30 mins by fluorescence based assay
- ChEMBL_1614246 (CHEMBL3856315) Non-competitive inhibition of recombinant yeast glutathione-S-transferase-tagged SIRT2 using acetylated histone as substrate measured after 30 mins by fluorimetric analysis
- Enzyme Inhibition Assay Purified 5-LO was added to reaction mix containing the test compounds. For efficient inhibition of 5-LO, low hydroperoxide levels are important. Glutathione (GSH) and bovine glutathione peroxidase-1 were included in the assay. The reaction was started by the addition of AA and CaCl2. After incubation, reaction was stopped with 1 ml of methanol, and formed 5-LO metabolites were extracted and analyzed by HPLC.
- ChEMBL_2071492 (CHEMBL4727026) Inhibition of recombinant human N-terminal Met and 6-His-tagged Glyoxalase-1 (Ala2 to Met184 residues) using methylglyoxal and reduced glutathione as substrates by spectrophotometric method
- ChEMBL_2237123 (CHEMBL5151019) Inhibition of NAE (unknown origin) mediated neddylation assessed as decrease in NEDD8-Ubcl2 adduct formation incubated for 2 hrs in presence of L-glutathione and ATP by HTRF assay
- ChEMBL_2199182 (CHEMBL5111698) Inhibition of human glutathione S-transferase-tagged LIMK1 (321 to 647 residues) expressed in baculovirus-infected Sf9 cells incubated for 30 mins by radiometric scintillation counting analysis
- ChEMBL_2199183 (CHEMBL5111699) Inhibition of human glutathione S-transferase-tagged LIMK2 (312 to 638 residues) expressed in baculovirus-infected Sf9 cells incubated for 60 mins by radiometric scintillation counting analysis
- ChEMBL_1463404 (CHEMBL3399741) Inhibition of NH2-terminal glutathione S-transferase fused human recombinant IGF-IR catalytic domain expressed in insect cells using poly(Glu:Tyr) substrate by ELISA-based assay
- ChEMBL_1462105 (CHEMBL3396812) Inhibition of mPGES-1 in interleukin-1beta-stimulated human A549 cells microsomal membranes assessed as reduction in PGE2 formation incubated for 15 mins in presence of PGH2 and glutathione by RP-HPLC method
- ChEMBL_1902456 (CHEMBL4404678) Inhibition of recombinant GSTO1-1 (unknown origin) expressed in Escherichia coli using S-(4-nitrophenacyl)glutathione as substrate preincubated for 2 mins followed by substrate addition by 4-NPG assay
- ChEMBL_2304438 Binding affinity to human H-PGDS expressed in Escherichia coli BL21 DE3 cells assessed as equilibrium dissociation constant by measuring changes in intrinsic tryptophan fluorescence in presence of glutathione by fluorescence based analysis
- ChEMBL_760655 (CHEMBL1815640) Inhibition of human MRP1 expressed in Spodoptera frugiperda Sf9 cells assessed as inhibition of NEM-GS-induced vanadate-sensitive ATPase activity measured by generation of inorganic phosphate by spectrophotometry in presence of glutathione
- ChEMBL_1648196 (CHEMBL3997252) Inhibition of human N-terminal His6-tagged GLO1 expressed in baculovirus infected sf21 cells assessed as reduction in S-D-lactoylglutathione formation measured for 5 mins in presence of glutathione by spectrophotometric method
- ChEMBL_940945 (CHEMBL2330635) Inhibition of ATR in human HeLa cell nuclear extracts using glutathione S-transferase-p53N66 and ATP as substrate incubated for 10 mins prior to ATP addition measured after 1 hr by ELISA
- ChEMBL_1989414 (CHEMBL4623149) Inhibition of recombinant human Glyoxalase-1 expressed in Escherichia coli BL21 (DE3) assessed as inhibition of S-D-lactoylglutathione formation using MG and reduced glutathione as substrate measured for 2 mins by spectrophotometric method
- ChEMBL_2203501 (CHEMBL5116209) Inhibition of mouse glutathione S-transferase-tagged Clk1 expressed in Escherichia coli DH5alpha using NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH peptide as substrate in presence of [gamma 32P-ATP] and ATP by liquid scintillation counting method
- ChEMBL_2203502 (CHEMBL5116210) Inhibition of mouse glutathione S-transferase-tagged Clk2 expressed in Escherichia coli DH5alpha using NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH peptide as substrate in presence of [gamma 32P-ATP] and ATP by liquid scintillation counting method
- ChEMBL_2203503 (CHEMBL5116211) Inhibition of mouse glutathione S-transferase-tagged Clk4 expressed in Escherichia coli DH5alpha using NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH peptide as substrate in presence of [gamma 32P-ATP] and ATP by liquid scintillation counting method
- ChEMBL_2203504 (CHEMBL5116212) Inhibition of mouse glutathione S-transferase-tagged Dyrk1A expressed in Escherichia coli DH5alpha using NH2-RSPSYGRSRSRSRSRSRSRSRSNSRSRSY-OH peptide as substrate in presence of [gamma 32P-ATP] and ATP by liquid scintillation counting method
- ChEMBL_1902458 (CHEMBL4404680) Inhibition of recombinant GSTO1-1 (unknown origin) expressed in Escherichia coli assessed as inhibitor constant using S-(4-nitrophenacyl)glutathione as substrate preincubated for 1 to 6 mins followed by substrate addition by spectrophotmetric analysis
- ChEMBL_1738015 (CHEMBL4153765) Inhibition of ATM derived from human HeLa cell nuclear extract using glutathione S-transferase p53N66 as substrate preincubated for 10 mins followed by ATP addition and subsequent incubation for 1 hr measured after 1.5 hrs by ELISA
- ChEMBL_2071491 (CHEMBL4727025) Inhibition of recombinant human N-terminal Met and 6-His-tagged Glyoxalase-1 (Ala2 to Met184 residues) using glutathione and methylglyoxal as substrates preincubated for 15 mins followed by enzyme addition by double beam UV-vis spectrophotometric method
- ChEMBL_1876789 (CHEMBL4378183) Inhibition of human N-terminal His6-tagged LTC4S expressed in Pichia pastoris X33 using LTA4 methyl ester and glutathione as substrate preincubated for 15 mins followed by substrate addition and measured after 30 mins by LC-MS/MS analysis
- Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50 Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Benjamin Cravatt, TSRI Network: Molecular Libraries Probe Production Centers Network (MLPCN) Grant Proposal Number: 1 R01 CA087660-05 Grant Proposal PI: Benjamin Cravatt, TSRI External Assay ID: GSTO1_INH_FLUO_ABPP_3XIC50 Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50. Description: Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide-variety of both exogenous and endogenous compounds for biotransformation and/or removal [1]. Using activity-based proteomic methods, we discovered that glutathione S-tranferase omega (GSTO1) is over-expressed in human cancer cell lines that show enhanced aggressiveness [2], and other studies have implicated GSTO1 in chemotherapeu
- Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50 Set 2 Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Benjamin Cravatt, TSRI Network: Molecular Libraries Probe Production Centers Network (MLPCN) Grant Proposal Number: 1 R01 CA087660-05 Grant Proposal PI: Benjamin Cravatt, TSRI External Assay ID: GSTO1_INH_FLUO_ABPP_3XIC50_SET2 Name: Late stage assay provider results from the probe development effort to identify inhibitors of GSTO1: Gel-based activity-based protein profiling (ABPP) IC50 Set 2. Description: Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide-variety of both exogenous and endogenous compounds for biotransformation and/or removal [1]. Using activity-based proteomic methods, we discovered that glutathione S-tranferase omega (GSTO1) is over-expressed in human cancer cell lines that show enhanced aggressiveness [2], and other studies have implicated GSTO1 in ch
- In Vitro GST Activity Assay The PGDS glutathione-S-transferase (GST) activity was measured by using MonoChloroBimane (MCB) as a chromogenic substrate. The assay was run at 384-well format in reaction buffer containing enzyme and test compound. Then MCB and GSH were added for further incubation. After reaction was stopped, the fluorescence was measured (excitation: 390 nm; emission: 478 nm).
- Glutathione S-transferase Activity Assay Enzymatic activity was determined spectrophotometrically by measuring the conjugation of CDNB with GSH. The 1 ml assay mixture contained 0.5 mM CDNB, 1 mMGSH, and 100 mM potassium phosphate buffer, pH 6.5. The rate of increase in absorbance at 340 nm was measured for 5 mm at 37°C against a blank containing thereaction mixture without enzyme.
- ACAT-1 Enzyme Inhibition Assay The assay mixture contains test compound, membrane protein, cholesterol solubilized in Triton WR-1339, and glutathione in a 96-deep well plate. The reaction was started by adding substrate [14C]oleoyl coenzyme A and incubated. After the reaction was stopped, radioactive cholesteryl oleate product was separated into the organic heptane phase. ACAT-1 activity was quantified by counting aliquots of the upper heptane layer by liquid scintillography.
- Glutathione Reductase Activity Assay Enzymatic activity was measured by Beutler's method with a Shimadzu Spectrophotometer UV-(1208), at 25°C. The assay system contained 100 mM Tris-HCl buffer pH 8.0, including 0.5 mM EDTA, 3.3 mM GSSG and 0.1 mM NADPH. One enzyme unit is defined as the oxidation of 1 μmol NADPH per min under the assay condition at 25°C.
- Binding Assay Stable and optimal assay conditions were determined by cross-titrating GST-AKAP18 δ and biotinylated PLB using 10 μg/ml glutathione acceptor beads and 10 μg/ml streptavidin donor beads in an AlphaScreen assay. The excitation wavelength was 680 nm, with the emission wavelength being 520-560 nm. Signal intensity in each well was registered and the optimal concentration to use was chosen to be the concentration prior to the peak of the signal.
- Binding Assay Stable and optimal assay conditions were determined by cross-titrating GST-AKAP18δ and biotinylated PLB using 10 μg/ml glutathione acceptor beads and 10 μg/ml streptavidin donor beads in an AlphaScreen assay. The excitation wavelength was 680 nm, with the emission wavelength being 520-560 nm. Signal intensity in each well was registered and the optimal concentration to use was chosen to be the concentration prior to the peak of the signal.
- PDGFR-beta Inhibition Assay The autophosphorylation assay was performed in 96-well plates using the recombinant GST-fused kinase domains expressed in baculovirus and purified over glutathione-Sepharose. 33P-ATP was used as the phosphate donor, and the PDGFR-beta was used as the acceptor. IC50 values were calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at three concentrations (usually, 0.01, 0.1, and 1 uM or 0.1, 1, and 10 uM).
- Kinase Inhibition Assay The in vitro kinase assays were performed in 96-well plates using the recombinant GST-fused kinase domains expressed in baculovirus and purified over glutathione-Sepharose. 33P-ATP was used as the phosphate donor, and the polyGluTyr (4:1) peptide was used as the acceptor. IC50 values were calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at three concentrations (usually, 0.01, 0.1, and 1 uM or 0.1, 1, and 10 uM).
- Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in the presence of Glutathione. The Cdc25B Phosphatase probe assessment dose response assay in Glutathione (GSH) has been developed to assess actives that were identified in the Cdc25B HTS AID 368 and confirmed in the Cdc25B 10-pt dose response confirmation assay AID 569 conducted by the University of Pittsburgh Molecular Screening Center (PMLSC) part of the Molecular Library Screening Center Network (MLSCN). XO1 submission MH078959 Cdc25B catalytic domain in vitro assay, Assay Provider Dr. Marni Brisson, Department of Pharmacology at the University of Pittsburgh. The 107 protein tyrosine phosphatases (PTPs) found in the human genome are defined by the active site sequence C(X)5R(S/T), with X being any amino acid, and they are critical regulators of mammalian cell proliferation, differentiation, and apoptosis. The active site cysteine of PTPs is required for catalytic activity and performs a nucleophilic attack on the phosphotyrosine residues of the substrate to form a covalent thiol-phosphate intermediate followed
- Kinase Assay Pharmacol 2002, 62 58-62) The compounds named in the specified Examples were tested as follows for inhibition of ALK-5 autophosphorylation activity and of the ALK-5 phosphorylation of α-Casem Materials. Buffer 50 mM HEPES, pH 76, with 10 mM NaCI, 10 mM MgCI2, and 1 mM DTT GST-ALK-5 protein - 0 44 mg/ml (roughly 7 μM stock) A 1 350 dilution gives a 20 nM stock, which translates to 2 nM final in assay Human ALK-5 was expressed in Sf9 insect cells infected with Bacul ovirus expressing a ALK-5 truncation sequence (amino acids H149 -M503), fused at the N-terminus to Glutathione S-transferase GST, in a pFastBac vector (Invitrogen) The cells were disrupted by sonication at 4 0C The lysate was centrifuged at 40,000 x g for 45 minutes, and the supernantant applied to a 10 ml column of Glutathione Sepharose 4 Fast Flow (AmershamBioscienses) equilibrated with 100 mM Tπs-HCI pH 76 buffer containing 300 mM NaCI, 10% glycerol, 1 % NP40, 2 mM dithiothreitol (DTT) and one Protease...
- hPrep Inhibition Assay Table 3: Compounds were tested in a biochemical inhibition assay using Prolyl endopeptidase, Prolyl Oligopeptidase (hPREP) enzyme at 0.6 nM FAC (R&D Systems, 4308-SE) and the substrate Z-Gly-Pro-amino-methylcoumarin (Bachem, I-1145) at 50 μM FAC. 384 Low volume black plates (Greiner #784076) were used. 4 μL, 1.2 nM enzyme solution (25 mM Tris HCl, 250 mM NaCl, 0.01% Triton X-100, 5 mM Glutathione, pH 7.5) was added to 40 nL compounds (in DMSO) at 10 CR, 3-fold dilution series from 50 μM FAC. Plates were incubated for 15 min at rt in dark. 4 μL, 100 μM substrate solution (25 mM Tris HCl, 250 mM NaCl, 0.01% Triton X-100, 5 mM Glutathione, pH 7.5) was added to each well. Plates were centrifuged at 1000 rpm and incubated for 20 min at rt in dark. The plates were read on a PHERAstar reader with excitation 340 nm and emission 460 nm. D
- TryR Enzyme Inhibition Assay Inhibition of TryR was carried out in 96-well plates using a Biotek Precision 2000 automated liquid handler. Reaction was initiated by addition of NADPH to the assay mixtures contained TryR , trypanothione disulfide substrate, and inhibitor (100 uM to 10 nM in 3-fold serial dilutions). The linear rate of thionitrobenzoate ion formation was monitored over 5 min in a Spectramax 340PC plate reader (Molecular Devices) at 412 nm. Raw data was processed using Microsoft Excel. Grafit 5.0 (Erithacus software) was used to fit the data to a three-parameter equation to determine IC50 values. IC50 determinations were carried out in triplicate for each compound and the mean weighted to standard error calculated.
- FRET Activity Assay Expression and purification of the FXR-LBD: An overnight preculture of a transformed E. coli strain was diluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an optical density of OD600=0.4-0.6. Gene expression was then induced by addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Per liter of original cell culture, cells were resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell debris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept slowly rotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelleted by centrifugation (2000×g, 15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl2 and 1M NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl2 and 80 mM glutathione added immediately prior to use as powder). The suspension was left rotating for 15 min at 4° C., the beads pelleted and eluted again with half the volume of elution buffer than the first time. The eluates were pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl2 as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-Page.
- In Vitro Enzyme Assay ATR for use in the in vitro enzyme assay was obtained from HeLa nuclear extract (CIL Biotech, Mons, Belgium) by immunoprecipitation with rabbit polyclonal antiserum raised to amino acids 400-480 of ATR (Tibbetts R S et al, 1999, Genes Dev. 13:152-157) contained in the following buffer (25 mM HEPES (pH7.4), 2 mM MgCl2, 250 mM NaCl, 0.5 mM EDTA, 0.1 mM Na3V04, 10% v/v glycerol, and 0.01% v/v Tween 20). ATR-antibody complexes were isolated from nuclear extract by incubating with protein A-Sepharose beads (Sigma, #P3476) for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, 10 ATR-containing Sepharose beads were incubated with 1 μg of substrate glutathione S-transferase-p53N66 (NH2-terminal 66 amino acids of p53 fused to glutathione {circumflex over ( )}-transferase was expressed in E. coli) in ATR assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 0.1 mM Na3V04, 0.1 mM DTT, and 10% (v/v) glycerol) at 37° C. in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP was added to a final concentration of 3 μM and the reaction continued at 37° C. for an additional 1 hour. The reaction was stopped by addition of IOOμ PBS and the reaction was transferred to a white opaque glutathione coated 96-well plate (NUNC #436033) and incubated overnight at 4° C. This plate was then washed with PBS/0.05%>(v/v) Tween 20, blotted dry, and analyzed by a standard ELISA (Enzyme-Linked Immunosorbent Assay) technique with a phospho-serine 15 p53 (16G78) antibody (Cell Signaling Technology, #9286).
- hPrep Inhibition Assay Table 3: Compounds were tested in a biochemical inhibition assay using Prolyl endopeptidase, Prolyl Oligopeptidase (hPREP) enzyme at 0.6 nM FAC (R&D Systems, 4308-SE) and the substrate Z-Gly-Pro-amino-methylcoumarin (Bachem, I-1145) at 50 μM FAC. 384 Low volume black plates (Greiner #784076) were used. 4 μL, 1.2 nM enzyme solution (25 mM Tris HCl, 250 mM NaCl, 0.01% Triton X-100, 5 mM Glutathione, pH 7.5) was added to 40 nL compounds (in DMSO) at 10 CR, 3 fold dilution series from 50 μM FAC. Plates were incubated for 15 min at rt in dark. 4 μL, 100 μM substrate solution (25 mM Tris HCl, 250 mM NaCl, 0.01% Triton X-100, 5 mM Glutathione, pH 7.5) was added to each well. Plates were centrifuged at 1000 rpm and incubated for 20 min at rt in dark. The plates were read on a PHERAstar reader with excitation 340 nm and emission 460 nm. Data were analyzed in Genedata Screener . IC50 values were determined by plotting % inhibition versus log compound concentration and using a one site dose response model.
- B-RAF (V600E) Kinase Inhibition Assay The biological activities (IC50s) of the compounds were determined against the B-RAF (V600E) mutant enzyme in vitro. GST-MEK1, B-RAF (V600E), and inhibitor were added to the wells of glutathione-coated plate, and the plate was preincubated for 10 min. ATP was added to each well, and the plates were incubated for 45 min. Primary antibody (rabbit anti-phospho MEK1/2) and Eu-labeled anti-rabbit secondary antibody were added to the plates. After incubation, DELFIA enhancement solution (Perkin-Elmer, Turku, Finland) was added. The europium counts were measured on a Victor 2 reader (Perkin-Elmer, Turku, Finland).
- IRAK4 Kinase Assay The IRAK4-inhibitory activity of the inventive substances was measured in the IRAK4 TR-FRET assay (TR-FRET=Time Resolved Fluorescence Resonance Energy Transfer) described hereinafter.Recombinant fusion protein from N-terminal GST (glutathione S-transferase) and human IRAK4, expressed in baculovirus-infected insect cells (Hi5, BTI-TN-5B1-4, cell line purchased from Invitrogen, catalogue No. B855-02) and purified via affinity chromatography, was used as enzyme. The substrate used for the kinase reaction was the biotinylated peptide biotin-Ahx-KKARFSRFAGSSPSQASFAEPG (C-terminus in amide form) which can be purchased, for example, from Biosyntan GmbH (Berlin-Buch).
- In Vitro TrkC Activity Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- Inhibition Assay Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP-002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at -80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- TrkC Activity Human TrkC, catalytic domain [456-825 (end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- TrkC Activity Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip™3000.
- FRET Activity Assay The human FXRalpha LBD was expressed in E. coli strain BL21(DE3) as an N-terminally GST tagged fusion protein. The DNA encoding the FXR ligand binding domain was cloned into vector pDEST15 (Invitrogen). Expression was under control of an IPTG inducible T7 promoter. The amino acid boundaries of the ligand binding domain were amino acids 187-472 of Database entry NM_005123 (RefSeq). Expression and purification of the FXR-LBD: An overnight preculture of a transformed E. coli strain was diluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an optical density of OD600=0.4-0.6. Gene expression was then induced by addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Per liter of original cell culture, cells were resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell debris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept slowly rotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelleted by centrifugation (2000× g, 15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl2 and IM NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl2 and 80 mM glutathione added immediately prior to use as powder). The suspension was left rotating for 15 min at 4° C., the beads pelleted and eluted again with half the volume of elution buffer than the first time. The eluates were pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl2 as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-Page.
- Competitive Assay The PI3 Kinase Activity/Inhibitor Assay is a competitive assay used for the fast and sensitive quantitation of activity of the four class I PI3 kinases (p110 α, β, γ, δ). The PI3 Kinase Activity/Inhibitor Assay works on the principle that PI3 Kinase phosphorylates P1(3,4)P2 (PIP2), converting it to PI(3,4,5)P3 (PIP3). The PH domain of the protein GRP-1 binds PIP3 with high affinity and specificity. The kit includes this recombinant protein that is used as the capture protein. This protein binds to the glutathione plate and captures either the PIP3 generated as part of the kinase reaction or the biotinylated-PIP3 tracer included in the kit. The captured biotinylated-PIP3 is detected using streptavidin-HRP conjugate and a colorimetric read out (OD 450). The lower the signal, the higher the PI3 Kinase activity.
- Fluorescence Polarization Binding Assay (FPBA) This assay essentially is available from Cayman Chemical Company as Catalog item #600007. The test data reported for the aforementioned Examples (H-PGDS FPBA IC.sub.50) were generated using a 96-well, instead of the 384-well, format.Detection analyte and H-PGDS-MBP fusion enzyme were incubated in the presence of reduced glutathione (5 mM) for 60-90 minutes at room temperature and FP was measured using a TECAN SAFIRE 2 plate reader equipped with absorbance, fluorescence, fluorescence polarization and FRET capabilities. Assays were performed in 96-well microtiter plates in 100 .mu.L of total sample volume. Excitation and emission wavelengths appropriate for the employed detection analyte were used.
- Inhibition Assay mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
- Spectrophotometric 384 Well Assay Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
- Inhibitory Activity Assay Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
- Cell-Free Axl Kinase Inhibitory Activity A kinase dilution solution containing 170 ng/ml AXL (the 464th to 885th amino acids of the intracellular domain of human AXL expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography; Carna Biosciences, Inc., catalog No. 08-107) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 30 minutes, a solution containing a substrate peptide (FL-Peptide 30 (5FAM-KKKKEEIYFFF-CONH2), Caliper Life Sciences, catalog No. 760430) and ATP at 1.5 μM and 10 μM, respectively, was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 1.5 hours, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
- Cell-Free Mer Kinase Inhibitory Activity A kinase dilution solution containing 20 ng/ml Mer (the 528th to 999th amino acids of the intracellular domain of human MER expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography and ion-exchange chromatography; Carna Biosciences, Inc., catalog No. 08-108) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 20 minutes, a solution containing a substrate peptide (FL-Peptide 27 (5FAM-EFPIYDFLPAKKK-CONH2), Caliper Life Sciences, catalog No. 760424) and ATP at 5 mM was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 45 minutes, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
- Kinase Inhibition Assay Measurement of the kinase inhibitory activity of each compound produced in Examples was conducted using the Off-chip Mobility Shift Assay. For this test, a human recombinant VEGF receptor 2 was prepared in a baculovirus expression system. A recombinant protein was expressed as a GST fusion protein by using 790-1356 amino acids of a cytosolic domain in the VEGF receptor 2 (NP 002244.1) and binding a glutathione-S-transferase (GST) to N-terminal thereof. The expressed GST-VEGF receptor 2 fusion protein was purified using glutathione-sepharose chromatography. In addition, the test substance was dissolved in dimethylsulfoxide to prepare a solution at a concentration about 100 times higher than the test concentration. Furthermore, the solution was diluted with an assay buffer (20 mM HEPES, 0.01% Triton X-100 and 2 mM DTT, pH7.5) by 25 times to prepare a 4-time concentrated test substance solution. In the kinase inhibition assay, CSKtide was used as a substrate. In the kinase reaction, 10 mL of 2-time concentrated VEGF receptor 2 kinase solution, 5 mL of 4-time concentrated test substance solution prepared with the assay buffer, and 5 mL of 4-time concentrated substrate/ATP/metal solution were mixed in wells of a polypropylene 384-well plate, and reacted at room temperature for 1 hour (substrate concentration: CSKtide 1000 nM, ATP concentration: 75 μM, Magnesium: 5 mM). One hour after, 60 mL of Termination Buffer (QuickScout Screening Assist MSA) was added so as to terminate the reaction. After that, the substrate peptide and the phosphorylated peptide in the reaction solution were separated by LabChip3000 system (Caliper Life Science), and the both peptides were quantified.
- Enzyme Assay The aim of this assay is to determine the affinity of a test compound for the mPGES-1 enzyme. 47 ul of recombinant human mPGES-1 (0.5 ug protein/well) containing microsomal suspension in a buffer containing GSH, (2.5 mmol/L L-Glutathione reduced, dissolved in 0.1 mol/L Phosphat Buffer pH 7.4) is dispensed in a 384-well plate and thereafter 1 ul of the test compound(s) is/are added and incubated for 25 minutes at room temperature. The enzyme reaction is started by the addition of 2 ul PGH2 (final conc. 2 uM) dissolved in water-free Diglyme. After 60 seconds the reaction is terminated by addition of a stop solution containing FeCl2 (10 uL 0.074 mol/l FeCl2). The samples are diluted between 1:25 in PBS (Phosphate Buffered Saline). 10 ul of the diluted samples are transferred to 384-well low volume plate.
- Inhibition Assay Reader: Synergy HT program: tyrosinase 280-490 kinetics: kinetics over 45 minutes, reading at t=10 minutes, Tests in transparent 96-well plates, Phosphate buffer (pH 6.8), Enzyme: mushroom tyrosinase (T-3824, Sigma), Substrate: L-tyrosine (T-3754, Sigma), Positive control: Kojic acid (KA) (60890, Fluka) (reference inhibitor). Reference Molecules for the Test: Kojic acid: 9 μM
- Factor XIIa 1536 HTS Dose Response Confirmation Molecular Library Screening Center Network (MLSCN) Penn Center for Molecular Discovery (PCMD) Assay Provider: Scott L. Diamond, University of Pennsylvania MLSCN Grant: X01-MH076406-01 Target Factor XII (FXII) is a 80 kDa zymogen found at a concentration of 0.375 uM in plasma, and upon activation by kallikrein at R353, a disulfide-linked two chain molecule called factor XIIa alpha (FXIIa) is generated. FXIIa is also capable of autoactivation by binding to negatively charged surfaces (1). Kallikrein can also cleave other scissile bonds in FXIIa alpha outside of the catalytic domain at R334, R343, and R353, generating FXIIa beta, a 30 kDa enzyme that is no longer able to bind to surfaces, and which activates prekallikrein (PK) to kallikrein, using high molecular weight kininogen (HK) as a cofactor (2, 3). FXIIa is irreversibly inhibited by C-1 inhibitor (C1INH), a 105 kDa plasma SERPIN (4-6). FXII, PK, HK, C1INH, and factor XI (FXI) have been traditionally placed within the i
- Factor XIIa Dose Response Confirmation Molecular Library Screening Center Network (MLSCN) Penn Center for Molecular Discovery (PCMD) Assay Provider: Scott L. Diamond, University of Pennsylvania MLSCN Grant: X01-MH076406-01 Target Factor XII (FXII) is a 80 kDa zymogen found at a concentration of 0.375 uM in plasma, and upon activation by kallikrein at R353, a disulfide-linked two chain molecule called factor XIIa alpha (FXIIa) is generated. FXIIa is also capable of autoactivation by binding to negatively charged surfaces (1). Kallikrein can also cleave other scissile bonds in FXIIa alpha outside of the catalytic domain at R334, R343, and R353, generating FXIIa beta, a 30 kDa enzyme that is no longer able to bind to surfaces, and which activates prekallikrein (PK) to kallikrein, using high molecular weight kininogen (HK) as a cofactor (2, 3). FXIIa is irreversibly inhibited by C-1 inhibitor (C1INH), a 105 kDa plasma SERPIN (4-6). FXII, PK, HK, C1INH, and factor XI (FXI) have been traditionally placed within the i
- EED A-395 Peptide Competition Assay For the assay, compounds or nonbiotinylated H3(23-34) peptide were dispensed in assay-ready plates using a three-fold serial dilution from 50 μM to ~850 pM using an Echo 550 Acoustic Liquid Handler (Labcyte). The following buffer was used to set up the binding reactions: 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.01% Tween-20, 10 μM DTT, and 0.05% BSA, with the DTT and BSA added fresh before initiating the binding assay. The binding assay was initiated by adding a 10 μl mixture of 1 nM GST-tagged EED, 15 nM biotinylated-histone H3 peptide, and 10 μg/ml each of the streptavidin donor and glutathione acceptor beads to the predispensed compounds. The reactions were then incubated for 1 h at 25 °C in a humidified chamber before detection on the PerkinElmer Envision plate reader using an AlphaLISA/Screen protocol.
- Enzymatic Assay The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 al of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 al of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, M nnedorf, Switzerland).
- Enzyme Assay The aim of this assay is to determine the affinity of a test compound for the mPGES-1 enzyme. 47 μl of recombinant human mPGES-1 (0.5 μg protein/well) containing microsomal suspension in a buffer containing GSH, (2.5 mmol/L L-Glutathione reduced, dissolved in 0.1 mol/L Phosphat Buffer pH 7.4) is dispensed in a 384-well plate and thereafter 1 μl of the test compound(s) is/are added and incubated for 25 minutes at room temperature. The enzyme reaction is started by the addition of 2 ul PGH2 (final conc 2 μM) disolved in water-free Diglyme. After 60 seconds the reaction is terminated by addition of a stop solution containing FeCl2 (10 μL 0.074 mol/l FeCl2). The samples are diluted between 1:25 in PBS (Phosphate Buffered Saline). 10 μl of the diluted samples are transferred to 384-well low volume plate.
- GLO1 Activity Assay Assays were carried out in 100 mM sodium phosphate, pH 7.0 buffer using 96-well Clear UV Plate (Corning UV Transparent Microplates; catalog #3635). A fresh solution of glutathione (pre-substrate 1, 100 mM) as well as methylglyoxal (pre-substrate 2, 100 mM) was prepared in deionized water. The substrate was prepared by adding 14.5 ml of buffer and 0.99 ml of each pre-substrate components. The substrate mixture was vortexed vigorously for 15 s, then allowed to sit at room temperature for 20 min. Initial well volume was 50 μl containing GLO1 (40 ng) and inhibitor. This protein and inhibitor mixture was incubated for 15–20 min before the addition of substrate. To this was then added substrate (150 μl), yielding a maximum amount of 5% DMSO per well. The enzyme activity was measured using a BioTek Synergy H4 plate reader by measuring absorbance at 240 nmevery 1 min for 8 min.
- Inhibition Assay To assess the inhibition of the MRP2, MRP3 and BSEP efflux transporters, an in vitro vesicular transporter assay from Solvo Biotechnology Inc. was used. The Test Articles (TAs) (at 0.068, 0.2, 0.62, 1.8, 5.5, 16.7 and 50 uM) were incubated with efflux transporter membrane vesicles (Solvo Biotechnology Inc.) both in the absence and presence of 4 mM ATP to distinguish between transporter mediated uptake and passive diffusion of TA's into the vesicles. In the case of MRP2 and MRP3 transporters reactions were carried out in the presence of 2 mM glutathione. Reaction mixtures were preincubated for ten minutes at 37 C. Reactions were started by the addition of 25 ul of 12 mM MgATP (4 mM final concentration in assay) or assay buffer for background controls. Reactions were stopped by adding 200 ul of ice-cold washing buffer and immediately followed by filtration on glass fiber filters in a 96-well format (filter plate).
- GRX Assay Human GRX-1 (0.25 unit/mL) was incubated at 25 °C with increasing concentrations of 2-AAPA (25, 50, 100, and 200 μM) and BSA (1 mg/mL). Aliquots were withdrawn for determination of GRX activity at 3, 10, and 20 min. Control incubations without 2-AAPA were conducted in parallel. The aliquots were added to the GRX assay solution, and enzyme activity was determined from a coupled reaction with GR based on a literature reported procedure with minor modification. In this assay, a mixed disulfide between GSH and the mercaptoethanol moiety derived from 2-hydroxyethyldisulfide (HED) served as the substrate for GRX; briefly GSH (10 mM) and HED (7 mM) were premixed in water for 5 min before transferring onto ice. The final GRXassay solution contained GSH (1 mM), HED (0.7mM), GR (0.02 units/mL), NADPH (0.2 mM), and bovine serum albumin (BSA, 1 mg/mL) in Trisbuffer (pH 8, 0.1 M). Theactivity was determined by monitoring the disappearance of NADPH spectrophotometrically at λ = 340 nm [Holmgren et al., Meth. Enzymol., 252:283-292].
- Biochemical Activity Assay TNKS 1 and 2: The activity ELISA is performed in 384 well Glutathione coated microtiter plates (Express capture Glutathione coated plate, Biocat, Heidelberg, Germany). The plates are pre-equilibrated with PBS. Then the plates are incubated with 50 μl 20 ng/well GST-tagged Tnks-1 (1023-1327 aa, prepared in-house), respectively GST-tagged Tnks-2 (873-1166 aa, prepared in-house) in assay buffer (50 mM HEPES, 4 mM Mg-chloride, 0.05% Pluronic F-68, 2 mM DTT, pH 7.7) overnight at 4° C. The plates are washed 3 times with PBS-Tween-20. The wells are blocked by incubation at room temperature for 20 minutes with 50 μl blocking buffer (PBS, 0.05% Tween-20, 0.5% BSA). Afterwards the plates are washed 3 times with PBS-Tween-20. The enzymatic reaction is performed in 50 μl reaction solution (50 mM HEPES, 4 mM Mg-chloride, 0.05% Pluronic F-68, 1.4 mM DTT, 0.5% DMSO, pH 7.7) with 10 μM bio-NAD (Biolog, Life science Inst., Bremen, Germany) as co-substrate in the absence or presence of the test compound (10 dilution concentrations) for 1 hour at 30° C. The reaction is stopped by 3 times washing with PBS-Tween-20. For the detection 50 μl of 20 ng/μl Streptavidin, HRP conjugate (MoBiTec, G ttingen, Germany) in PBS/0.05% Tween-20/0.01% BSA are added and the plates are incubated for 30 minutes at room temperature. After three times washing with PBS-Tween-20 50 μl of SuperSignal ELISA Femto Maximum sensitivity substrate solution (ThermoFisherScientific (Pierce), Bonn, Germany) are added. Following a 1 minute incubation at room temperature luminescence signals are measured with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at 700 nm.
- AlphaScreen Assay BRD4: 2.5 nM of BRD4(49-170) and 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
- AlphaScreen Assay CBP: 5 nM GST-CBP(1081-1197) and 20 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
- AlphaScreen Assay When two proteins interact (one of which is conjugated to the donor bead and the other to the acceptor bead) the beads are brought into close proximity. Excitation of a photosensitizer in the donor bead results in the production of singlet oxygen that diffuses to and reacts with a chemiluminescent molecule in the acceptor bead. This results in the activation of fluorophores in the acceptor bead which results emitted light that can be detected. In this assay, GST-MDM2 (1-150) and full-length His6-wt-p53 were expressed in E. coli and affinity purified under non-denaturing conditions. To detect the MDM2-p53 interaction by the AlphaScreen assay (Perkin Elmer), GST-MDM2, His6-p53, and potential inhibitors were mixed in binding buffer, and incubated for 1 h at 24 deg C. Nickel acceptor beads and glutathione donor beads were added. Following 1 h incubation at 24 deg C, the mixture was analyzed in a fluorometer at an excitation wavelength of 680 nm. Nutlin-3a obtained from Cayman Chemical was used as a control. The IC50 values reported are from runs repeated four times.
- Biochemical Assay A solution of test compound was added to a diluted microsome preparation containing human mPGES-1 and pre-incubated for 15 minutes in potassium phosphate buffer pH 6.8 with cofactor glutathione (GSH). Corresponding solutions without test compound were used as positive controls, and corresponding solutions without test compound and without microsomes were used as negative controls. The enzymatic reaction was then started by addition of the substrate PGH2 in an organic solution (dry acetonitrile).The typical reaction conditions of the enzymatic reaction were thus: Test compound: ranging from 60 uM to 0.002 uM, or zero in positive and negative controls; potassium phosphate buffer pH 6.8: 50 mM; GSH: 2.5 mM; mPGES-1-containing microsomes: 2 ug/mL (sample and positive controls) or 0 ug/mL (negative control); PGH2: 10.8 uM; Acetonitrile: 7.7% (v/v); DMSO: 0.6% (v/v). The reaction was stopped after one minute by adding an acidic solution (pH 1.9) of ferric chloride.
- CBP AlphaScreen Assay 5 nM GST-CBP(1081-1197) and 20 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) were incubated with varying concentrations of CBP inhibitors in 15 μL of buffer containing 50 mM HEPES 7.5, 100 nM NaCl, 1 mM TCEP, and 0.003% Tween-20. After 30 minutes incubation at room temperature, 15 μL of detection buffer (BPS Bio. 33006) containing 7 μg/mL of Glutathione AlphaLisa acceptor beads (Perkin Elmer AL109) and 14 μg/mL of Streptavidin donor beads (Perkin Elmer 676002) was then added to the previous mixture. The reaction was incubated for an additional 2 hours at room temperature, and the AlphaScreen signal was quantified using the Envision Multilabel plate reader. As negative control, GST-CBP(1081-1197) was incubated with the non-acetylated biotin-H4(1-21) peptide(AnaSpec. 62555) and in presence of 0.25% of final DMSO concentration.
- Coupled Nucleotide Exchange Assay (20 hours) Purified GDP-bound KRAS protein (aa 1-169), containing either G12C or G12D, as well as C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for 20 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay (5min) Purified GDP-bound KRAS protein (aa 1-169), containing either G12C or G12D, as well as C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay Purified GDP-bound KRAS protein (aa 1-169), containing both G12D and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- In Vitro Assay A PCR product covering residues 1058-1365 of c-Met (c-Met kinase domain) is generated from Human Liver QuickClone cDNA (Invitrogen) using forward primer 5'-ATTGACGGATCCATGCTAAATCCAGAGCTGGTCCAGGCA-3' (SEQ ID NO. 1) and reverse primer 5'-ACAACAGAATTCAATACGGAGCGACACATTTTACGTT-3' (SEQ ID NO. 2). The PCR product is cloned into a modified pFastBac 1 expression vector (harboring the gene for S. japonicum glutathione S-transferase immediately upstream of the multiple cloning site) using standard molecular biological techniques. The GST-c-Met kinase domain fusion (GST-Met) gene is transposed into full-length baculovirus DNA using the BacToBac system (Invitrogen). High5 cells are infected with the recombinant baculovirus for 72 h at 27 C. The infected cells are harvested by centrifugation and the pellet is stored at -80 C. The pellet is resuspended in buffer A (50 mM HEPES, pH 8.0, 0.25 M NaCl, 10 mM 2-mercaptoethanol, 10% (w/v) glycerol, 0.5% (v/v) protease.
- Inhibition Assay The test was carried out according to the method of Urade, Y. et al. (J. Biol. Chem., 262, 3820-3825, (1987)). More specifically, the reaction mixture (49 uL) containing 100 mM Tris-HCl (pH 8.0), 1 mM reduced glutathione, 0.1 mg/mL gamma-globulin, and human H-PGDS (q.s.), and a compound (final concentration: 0.01-100 uM) was preincubated at 25 C. for 5 minutes. Note that a DMSO solution (final concentration: 1%) was added to the solvent control group. Subsequently, 1 uL of [14C] prostaglandin H2 (final concentration: 10 uM) was added to start the reaction. One minute after the start of the reaction, 250 uL of a reaction stopper solution (diethylether/methanol/1 M citric acid (30/4/1) at a temperature of -20 C. was added to stop the reaction. After the reaction was stopped, 50 uL of the upper layer portion (organic solvent phase) was applied to a TLC plate and developed at -20 C. for 45 minutes.
- Spectrophotometric 384 Well Assay Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
- Spectrophotometric 384 Well Assay Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetylCoenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
- Spectrophotometric 384 Well Assay Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction.For biological testing, a human ACC2 construct which lacks the 128 amino acids at the N-terminus for increased solubility (nt 385-6966 in Genbank entry AJ575592) is cloned. The protein is then expressed in insect cells using a baculoviral expression system. Protein purification is performed by anion exchange.All compounds are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM.Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/mL BSA, 3.75 mM reduced L-glutathione, 15 U/mL lactate dehydrogenase, 0.5 mM phosphoenolpyruvate.
- Spectrophotometric 384 Well Assay Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction.For biological testing, a human ACC2 construct which lacks the 128 amino acids at the N-terminus for increased solubility (nt 385-6966 in Genbank entry AJ575592) is cloned. The protein is then expressed in insect cells using a baculoviral expression system. Protein purification is performed by anion exchange.All compounds are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM.Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate.
- CONFA Assay Confirmatory Assay (CONFA) used as a secondary screen for confirming the mechanism of action of compounds identified as hits using the ALA assay. The assay is performed using a similar procedure as ALA except that it utilizes SDS-PAGE for visualization and quantification of tau monomer and aggregated species (dimer, trimer, tetramer, pentamer, etc.). Only a single construct of tau without epitope tags is necessary, but the assay can use the same target prepared for the ALA assay also.For the CONFA assay, Tau target (300 nM) was prepared in buffer (Tris-HCl pH 7.4) and was incubated at room temperature for 3 hours with vehicle control (DMSO) and a dose range of a compound of the disclosure (0.098 uM-50 uM). Samples were mixed with an equal volume of 2×SDS sample buffer (4% SDS, 20% glycerol, 0.004% bromphenol blue, 125 mM Tris HCl, pH 7.0) to resolve tau monomer and disulfide-linked oligomers on 4-20% gradient polyacrylamide gels (Biorad) along with positive control (non-oligomerized tau target). The gels were stained with Oriole Fluorescent Gel Stain (BioRad) and imaged using the FluorChem R system (Protein Simple). AlphaView software (Protein Simple) was used for quantification of tau monomer and oligomers.
- Confirmatory Assay (CONFA) Confirmatory Assay (CONFA) used as a secondary screen for confirming the mechanism of action of compounds identified as hits using the ALA assay. The assay is performed using a similar procedure as ALA except that it utilizes SDS-PAGE for visualization and quantification of tau monomer and aggregated species (dimer, trimer, tetramer, pentamer, etc.). Only a single construct of tau without epitope tags is necessary, but the assay can use the same target prepared for the ALA assay also. For the CONFA assay, Tau target (300 nM) was prepared in buffer (Tris-HCl pH 7.4) and was incubated at room temperature for 3 hours with vehicle control (DMSO) and a dose range of a compound of the disclosure (0.098 uM-50 uM). Samples were mixed with an equal volume of 2×SDS sample buffer (4% SDS, 20% glycerol, 0.004% bromphenol blue, 125 mM Tris HCl, pH 7.0) to resolve tau monomer and disulfide-linked oligomers on 4-20% gradient polyacrylamide gels (Biorad) along with positive control (non-oligomerized tau target). The gels were stained with Oriole Fluorescent Gel Stain (BioRad) and imaged using the FluorChem R system (Protein Simple). AlphaView software (Protein Simple) was used for quantification of tau monomer and oligomers.
- KRAS::SOS1 AlphaScreen Binding Assay This assay is used to examine the potency with which compounds inhibit the protein protein interaction between SOS1 and KRAS G12D in a defined biochemical setting. Low IC50 values of given compounds are indicative of high potency of the SOS1 inhibitor compounds in this assay setting.Reagents: GST-TEV-SOS1 (564-1049) and His-TEV-Avi-KRAS G12D (1-169) are purchased from Viva Biotech (Shanghai) Ltd. GDP (Sigma, Cat. G7127) AlphaLISA Glutathione Acceptor Beads (PerkinElmer, Cat. AL109C) AlphaScreen Streptavidin Donor Beads (PerkinElmer, Cat. 6760002S) Assay plates: ProxiPlate-384 Plus, White 384-shallow well Microplate (PerkinElmer, Cat. 6008280)Assay Buffer: PBS, pH 7.4 (Gibco, Cat. 10010023) 0.05% Tween 20 (Sigma, Cat. P7949 100 ML) 0.1% Bovine Serum Albumin (BSA) (Sigma, Cat. A1933 5 G)Assay Protocol:SOS1 inhibitor compounds are diluted to a final start concentration of 1 PM. Serial dilutions of compounds are made using Tecan D300e Digital Dispenser in 9 concentrations with serial 1:3 dilutions. 100 nL of compound solution is transferred to the 384-well assay plate per well, covering a range between 1 μM and 0.15 nM minimum in duplicate. 10 nM (final assay concentration) KRAS G12D, 5 nM (final assay concentration) SOS1 and 10 μM (final assay concentration) GDP are mixed in assay buffer, and 5 μL of KRAS SOS1 GDP mix is added into the assay plate to the 100 nL of compound solution (final dilution in the assay 1:100, final DMSO concentration 1%). After a 30 min incubation, AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads are mixed in assay buffer at a concentration of 5 g/mL (final assay concentration), and 5 μL of bead mix is added into the assay plate. Plates are kept at room temperature in a darkened incubator for 3 h. After a 3 h incubation, the signal is determined using Envision (PerkinElmer). The excitation wavelength is 680 nm, and emission 615 nm. IC50 values are calculated and analyzed using GraphPad Prism.
- MKNK1 TR-FRET Assay A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany). For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 uL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 uL of a solution of adenosine-tri-phosphate (ATP, 16.7 uM=>final conc. in the 5 uL assay volume is 10 uM) and substrate (0.1 uM=>final conc. in the 5 uL assay volume is 0.06 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 ug/ml. The reaction was stopped by the addition of 5 uL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
- FRET Activity Assay Determination of a ligand mediated cofactor peptide interaction to quantify ligand binding to the nuclear receptor FXR was performed as follows.Preparation of human FXR alpha ligand binding domain: The human FXRalpha LBD was expressed in E. coli strain BL21(DE3) as an N-terminally GST tagged fusion protein. The DNA encoding the FXR ligand binding domain was cloned into vector pDEST15 (Invitrogen). Expression was under control of an IPTG inducible T7 promoter. The amino acid boundaries of the ligand binding domain were amino acids 187-472 of Database entry NM_005123 (RefSeq). Expression and purification of the FXR-LBD: An overnight preculture of a transformed E. coli strain was diluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an optical density of OD600=0.4-0.6. Gene expression was then induced by addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Per liter of original cell culture, cells were resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell debris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept slowly rotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelleted by centrifugation (2000×g, 15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl2 and 1M NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl2 and 80 mM glutathione added immediately prior to use as powder). The suspension was left rotating for 15 min at 4° C., the beads pelleted and eluted again with half the volume of elution buffer than the first time. The eluates were pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl2 as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-Page.The method measures the ability of putative ligands to modulate the interaction between the purified bacterial expressed FXR ligand binding domain (LBD) and a synthetic biotinylated peptide based on residues 676-700 of SRC-1 (LCD2, 676-700). The sequence of the peptide used was B-CPSSHSSLTERHKILHRLLQEGSPS-COOH (SEQ ID NO: 1) where the N-terminus was biotinylated (B). The ligand binding domain (LBD) of FXR was expressed as fusion protein with GST in BL-21 cells using the vector pDEST15. Cells were lysed by sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. For screening of compounds for their influence on the FXR-peptide interaction, the Perkin Elmer LANCE technology was applied. This method relies on the binding dependent energy transfer from a donor to an acceptor fluorophor attached to the binding partner of interest. For ease of handling and reduction of background from compound fluorescence LANCE technology makes use of generic fluorophore labels and time resolved detection Assays were done in a final volume of 25 μL in a 384 well plate, in a Tris-based buffer (20 mM Tris-HCl pH 7.5; 60 mM KCl, 5 mM MgCl2; 35 ng/μL BSA), containing 20-60 ng/well recombinantly expressed FXR-LBD fused to GST, 200-600 nM N-terminally biotinylated peptide, representing SRC1 aminoacids 676-700, 200 ng/well Streptavidin-xlAPC conjugate (Prozyme) and 6-10 ng/well Eu W1024-antiGST (Perkin Elmer). DMSO content of the samples was kept at 1%. After generation of the assay mix and diluting the potentially FXR modulating ligands, the assay was equilibrated for 1 h in the dark at rt in FIA-plates black 384 well (Greiner). The LANCE signal was detected by a Perkin Elmer VICTOR2VTM Multilabel Counter.
- RET Activity Assay Preparation of human FXR alpha ligand binding domain: The human FXRalpha LBD was expressed in E. coli strain BL21(DE3) as an N-terminally GST tagged fusion protein. The DNA encoding the FXR ligand binding domain was cloned into vector pDEST15 (Invitrogen). Expression was under control of an IPTG inducible T7 promoter. The amino acid boundaries of the ligand binding domain were amino acids 187-472 of Database entry NM_005123 (RefSeq). Expression and purification of the FXR-LBD: An overnight preculture of a transformed E. coli strain was diluted 1:20 in LB-Ampicillin medium and grown at 30° C. to an optical density of OD600=0.4-0.6. Gene expression was then induced by addition of 0.5 mM IPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cells were collected by centrifugation (7000×g, 7 min, rt). Per liter of original cell culture, cells were resuspended in 10 mL lysis buffer (50 mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and left on ice for 30 min. Cells were then subjected to sonication and cell debris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL of supernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen) was added and the suspension kept slowly rotating for 1 h at 4° C. Glutathione 4B sepharose beads were pelleted by centrifugation (2000×g, 15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4 mM MgCl2 and 1M NaCl). The pellet was resuspended in 3 mL elution buffer per liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5 mM MgCl2 and 80 mM glutathione added immediately prior to use as powder). The suspension was left rotating for 15 min at 4° C., the beads pelleted and eluted again with half the volume of elution buffer than the first time. The eluates were pooled and dialysed overnight in 20 mM Hepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl2 as well as 1 mM dithiothreitol and 10% (v/v) glycerol. The protein was analysed by SDS-Page.The method measures the ability of putative ligands to modulate the interaction between the purified bacterial expressed FXR ligand binding domain (LBD) and a synthetic biotinylated peptide based on residues 676-700 of SRC-1 (LCD2, 676-700). The sequence of the peptide used was B-CPSSHSSLTERHKILHRLLQEGSPS-COOH (SEQ ID NO: 1) where the N-terminus was biotinylated (B). The ligand binding domain (LBD) of FXR was expressed as fusion protein with GST in BL-21 cells using the vector pDEST15. Cells were lysed by sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. For screening of compounds for their influence on the FXR-peptide interaction, the Perkin Elmer LANCE technology was applied. This method relies on the binding dependent energy transfer from a donor to an acceptor fluorophor attached to the binding partner of interest. For ease of handling and reduction of background from compound fluorescence LANCE technology makes use of generic fluorophore labels and time resolved detection Assays were done in a final volume of 25 μL in a 384 well plate, in a Tris-based buffer (20 mM Tris-HCl pH 7.5; 60 mM KCl, 5 mM MgCl2; 35 ng/μL BSA), containing 20-60 ng/well recombinantly expressed FXR-LBD fused to GST, 200-600 nM N-terminally biotinylated peptide, representing SRC1 aminoacids 676-700, 200 ng/well Streptavidin-xlAPC conjugate (Prozyme) and 6-10 ng/well Eu W1024-antiGST (Perkin Elmer). DMSO content of the samples was kept at 1%. After generation of the assay mix and diluting the potentially FXR modulating ligands, the assay was equilibrated for 1 h in the dark at rt in FIA-plates black 384 well (Greiner). The LANCE signal was detected by a Perkin Elmer VICTOR2V Multilabel Counter. The results were visualized by plotting the ratio between the emitted light at 665 and 615 nm. A basal level of FXR-peptide formation is observed in the absence of added ligand.
- AlphaScreen Assay Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 al of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 jag/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
- AlphaScreen Assay Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
- AlphaScreen Assay Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15nM GST-BRD4-BD1 protein (aa 44-168) or 150nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
- AlphaScreen Assay The following example describes an assay that measured the ability of compounds to inhibit the binding of p53 to MDM2 using the AlphaScreen assay technology (PerkinElmer). The following protocol is an adaptation of the method described by H. R. Lawrence et al. (Bioorg. Med. Chem. Lett. 19 (2009) 3756-3759). Recombinant, truncated, human, N-terminal GST-MDM2 (aa 1-150) was obtained from GeneScript. Wild-type, full length human N-terminal 6-his p53 was purchased from SignalChem.Resulting in a final reaction volume of 24 μl PBS, 0.1% Tween-20, and 10% glycerol, 30 ng of MDM2 was added, followed by the addition of 10 of compound diluted in 100% DMSO that provided a final DMSO concentration of 4%. 30 ng of p53 was then added, mixed, and incubated at room temperature for 1 hour. Glutathione donor beads and Nickel acceptor beads (0.5 μg each; PerkinElmer) were added under subdued lighting conditions to a final reaction volume of 30 μl/well in a 96 well, volume Proxima plate.
- Biochemical Assay The following assay conditions were employed: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated with a compound dose-response titration for 5 min in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 5 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- DELFIA Assay The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
- DELFIA assay The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates. 10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 uL of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4).
- Dose response confirmation of DNMT1 inhibitors in a Fluorescent Molecular Beacon assay Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA) Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN) Grant Proposal Number: 1 R03 DA031091-01A1 Assay Provider: Dr. James Stivers, Johns Hopkins University, Baltimore, MD Hypermethylation of CpG dinucleotides in promoter regions of tumor suppressor genes by DNA 5-C-MTases is an important hallmark of human cancers [1-3]. Such reversible epigenetic silencing is a key pathway resulting in loss-of-function phenotypes that promote the growth of many cancers. Examples of genes that have undergone hypermethylation-induced silencing include the genes encoding the cell cycle regulator proteins pIS and p16, the pro-apoptotic effector gene Apaf-1, the mismatch DNA repair gene MLH1, and GSTP1 that codes for the phase 2 enzyme glutathione S-transferase [1, 2, 4-9]. Thus, inhibitors of MTase enzymes have the demonstrated potent
- In Vitro hLTC4 Synthase Enzyme Assay (Test A) In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 is converted to LTC4. Recombinant human LTC4 synthase is expressed in Piccia pastoralis and the purified enzyme is dissolved in 25 mM tris-buffer pH 7.8 supplemented with 0.1 mM glutathione (GSH) and stored at −80 °C. In order to obtain IC50-values for the compounds, the following procedure was used: A volume (24 μL) of 0.25 g/mL LTC4 synthase in 75 mM tris (pH ~8.5), 0.5 mM MgCl2 and 3 mM GSH was preincubated for 10 min with 0.5 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10^−9,5-10^−5 M as well as with DMSO only. The buffer is used as background. Runs are performed in duplicates. The enzymatic reaction is initiated by addition of 0.5 μL LTA4 in diglyme (final assay concentration 8 μM). The reaction is stopped after 1 min by addition of double the reaction volume of a stop solution (MeOH:H2O:acetic acid 70:30:1).
- Inhibiting Action The test was carried out according to the method of Urade, Y. et al. (J. Biol. Chem., 262, 3820-3825, (1987)). More specifically, the reaction mixture (49 μL) containing 100 mM Tris-HCl (pH 8.0), 1 mM reduced glutathione, 0.1 mg/mL γ-globulin, and human H-PGDS (q.s.), and a compound (final concentration: 0.01-100 μM) was preincubated at 25C. for 5 minutes. A DMSO solution (final concentration: 1%) was added to the solvent control group. Subsequently, 1 μL of [14C] prostaglandin H2 (final concentration: 10 μM) was added to start the reaction. One minute after the start of the reaction, 250 μL of a reaction stop solution (diethylether/methanol/1 M citric acid (30/4/1) at a temperature of −20C. was added to stop the reaction. After the reaction was stopped, 50 μL of the upper-layer portion (organic solvent layer) was applied to a TLC plate and developed at −20C. for 45 minutes (developing solvent: diethylether/methanol/acetic acid (90/2/1)).
- Inhibition Assay mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC). The solution free of test compound was used as positive control, and the solution free of test compound and microsome sample was used as negative control. 100% activity was defined as PGE2 production in the positive control minus PGE2 production in negative control. IC50 value was calculated by standard method.
- PI3K Inhibition Assay PI3K inhibition assay (PI3K Assay (Emmanuelle M, Huang Y, Yan H G et al. Targeting Protein Translation in Human Non-Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression. Cancer Res 67:(23). (2007).) was carried out by PI3 Kinase activity/inhibitor assay kit, where PI3 kinase reaction was set up in Glutathione-coated strips/plate for inhibitor reaction. Kinase and inhibitors were pre-incubated for 10 minutes prior to the addition of PIP2 substrate. 5 uL of 5x kinase reaction buffer were added in each well followed by the further addition of 5 uL/well of PIP2 substrate. Then distilled H2O was added to each well so as to make up a final volume of 25 uL/well. Incubation was done at rt for 1 hour which was followed by washing the wells 3 times with 200 uL of 1xTBST per well and then 2 times with 200 uL of IX TBS per well. Then 100 uL of the Substrate TMB per well was added and then to keep for colour development in the dark.
- RIP1 kinase assay RIP1 kinase assays were performed using ADP-Glo assay (Promega) according to manufacturer's protocol. Reactions were performed in 50 mM HEPES, pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.05% bovine serum albumin (BSA), 0.02% CHAPS buffer, containing 20 ng recombinant GST-human RIP1 kinase domain (amino acids 1-327), 50 mM ATP and 10 serial dilutions of inhibitors. Recombinant GST-RIP1 was generated using baculoviral expression system in Sf9 cells. Protein was purified by glutathione affinity chromatography, followed by size exclusion chromatography. Reactions were performed for 4 hr at room temperature and stopped by incubation with ADP-Glo reagent for 40 min at room temperature. Luminescent signal was developed by incubation with Kinase Detection reagent for 30 min at room temperature. Signal was determined using Victor3V platereader (Perkin Elmer). Non-linear regression to calculate EC50 values was performed using GraphPad Prism software package.
- SCD1 Enzymatic Assay The SCD1 enzymatic assay was done in a volume of 50 uL using 10 ug of RLM (prepared as described above) in a 96-well polypropylene plate (enzyme reaction buffer contains 0.1 M K-Phosphate Buffer, 10 mM ATP, 6 mM MgCl2, 1 mM CoA, 1 mM β-NADH, 1.6 mM L-glutathione, 20 uM Stearoyl-CoA). Stearoyl-[9,10-3H]-CoA (ARC-0390, 1 mCi/mL, 60 Ci/mmol,) was added at a final concentration of 2 uCi/mL. Test compound was then added to the reaction mixture at the selected concentration. After incubation at room temperature for 2 hours, 5 uL 1 N HCl was added to stop the reaction, followed by addition of 25 uL of 10% charcoal. The reaction mixture was then transferred to 96-well Multiscreen plate (Millipore, Cat# MSFCN6B50). [3H2O] was collected into Opti-plate (PE, Cat #6005290) by centrifuge, at 1,000 rpm for 2 minutes. 150 uL Microscint 40 (PE, cat #6013641) was then added to each well and counted on Topcount for [3H] counts per minute (cpm).
- Scintillation Proximity Assay (SPA The scintillation proximity assay (SPA) was run in white polystyrene flat-bottom 384-well plates (Greiner, cat. No. 781075). Assays were carried out in 40 i1 reaction volumes. Various concentrations of test ligands in 0.4 microlitres of DMSO were added to assay plates using an acoustic liquid dispenser. 4 nM purified N (HN)6-GST-TCS-hROR (258-518) was mixed with 40 micrograms Yttrium oxide (YOx) glutathione SPA imaging beads in assay buffer (20 mM Tris, 150 mM NaCl, 10% Glycerol, 0.25% CHAPS, 1 mM TCEP) prior to adding 30 microlitres to test ligands. Assay plates were incubated for one hour at room temperature before adding 10 microlitres tritiated 2-(4-(ethylsulfonyl)phenyl)-N-(4-(2-(methoxymethyl)phenyl)thiophen-2-yl)acetamide to test plates in assay buffer (final concentration, 25 nM). Test plates were incubated for 16 hours and read using a LEADseeker Multimodality imaging instrument.
- Kinase Assay A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein 56 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
- MKNK1 Kinase High ATP Assay TBDA recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
- AlphaScreen Binding Assay This assay can be used to examine the potency with which compounds inhibit the protein-protein interaction between SOS1 and KRAS G12D. This demonstrates the molecular mode of action of compounds. Low IC50 values are indicative of high potency of the SOS1 inhibitor compound in this assay setting:Reagents:GST-tagged SOS1 (564_1049_GST_TEV_ECO) produced in-houseGST-TEV-SOS1 (564-1049) is purchased from Viva Biotech Ltd.6??His-Tev-K-RasG12D(1-169)Avi is purchased from Xtal BioStructures, Inc. (Lot #X129-110)GDP (Sigma Cat No G7127)AlphaLISA Glutathione Acceptor Beads (PerkinElmer, Cat No AL109)AlphaScreen Streptavidin Donor Beads (PerkinElmer Cat No 6760002)Assay plates: Proxiplate-384 PLUS, white (PerkinElmer, Cat No 6008289)Assay Buffer:1??PBS0.1% BSA100 uM EDTA or without EDTA (IC50s in the tables are measured without EDTA unless they are marked with an asterisk)0.05% Tween 20KRAS SOS1 GDP Mix:10 nM (final assay concentration) KRAS G12D, 10 uM (final assay concentration) GDP and 5 nM (final assay concentration) GST-SOS1 are mixed in assay buffer prior to use and kept at room temperature.
- Coupled Nucleotide Exchange Assay (2 hours) Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay (20 hours) Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for 20 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay (5 minutes) Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 minutes. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 2 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Coupled Nucleotide Exchange Assay Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with serially diluted compound for either 2 h or 20 h. For all subsequent steps, DTT was added to the reaction buffer at a final concentration of 1 mM. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 5 min. The assay plates were then read on a plate reader measuring luminescence signal. Signal intensity of compound-containing wells were normalized to DMSO control, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
- Enzymatic Assay The SCD1 enzymatic assay was done in a volume of 50 μL using 10 μg of RLM (prepared as described above) in a 96-well polypropylene plate (enzyme reaction buffer contains 0.1 M K-Phosphate Buffer, 10 mM ATP, 6 mM MgCl2. 1 mM CoA, 1 mM β-NADH, 1.6 mM L-glutathione, 20 μM Stearoyl-CoA). Stearoyl-[9,10-3H]-CoA (ARC-0390, 1 mCi/mL, 60 Ci/mmol,) was added at a final concentration of 2 μCi/mL. Test compound was then added to the reaction mixture at the selected concentration. After incubation at room temperature for 2 hours, 5 μL 1 N HCl was added to stop the reaction, followed by addition of 25 μL of 10% charcoal. The reaction mixture was then transferred to 96-well Multiscreen plate (Millipore, Cat#MSFCN6B50). [3H2O] was collected into Opti-plate (PE, Cat#6005290) by centrifuge, at 1,000 rpm for 2 minutes. 150 μL Microscint 40 (PE, cat #6013641) was then added to each well and counted on Topcount for [3H] counts per minute (cpm).
- Evaluation of ABHD6 Enzyme Inhibitory Activity First, 1-arachidonoyl glycerol (Cayman Chemical) as a substrate was prepared with an assay buffer containing 50 mM tris-HCl (pH 7.4), 100 mM NaCl, and 0.05% BSA, so as to have a final concentration of 10 μmol/L. Then, a compound was added therein so as to have a final concentration of 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, or 10 μmol/L (DMSO: final concentration of 0.3%). In addition, solutions to which DMSO was added so as to have a final concentration of 0.3% were prepared as a group to which the compound was not added. The enzyme reaction was started by adding recombinant human ABHD6 (33-337) prepared with the same assay buffer to a mixed solution of the substrate and the compound so as to have a final concentration of 300 μg/mL. The recombinant human ABHD6 (33-337) was a GST-tagged ABHD6, and one which was expressed in E. coli, then purified with a Glutathione-Sepharose 4B resin, and then concentrated was used. The enzyme reaction was carried out at room temperature using a 384-well microplate made of polypropylene, and wells to which no enzyme was added were designated as a blank group.
- Homogenous Time-Resolved Fluorescence Assay (HTRF) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture.
- Human LXRalpha and LXRbeta Coactivator Recruitment FRET Assay This assay is based on the ability of the LXR-LBDs (LXRα and LXRβ) to recruit and interact with a co-activator peptide. This assay was run in agonist mode, to characterize compounds of the invention that exhibit LXR agonist activity, and in antagonist mode to characterize compounds of the invention having LXR antagonist activity and which cause a concentration-dependent release of the co-activator peptide in the presence of the full LXR agonist T0901317. The interaction between glutathione S-transferase (GST)-tagged, recombinant human LXRα-LBD or LXRβ-LBD and fluorescein-conjugated co-activator peptide SRC2-3 (Cat# PV4588, Life Technologies, Grand Island, N.Y.) was measured via time resolved-fluorescence resonance energy transfer (TR-FRET). The assay was performed in 96-well half-area black Opti-plates (Cat# 3686, Corning, Lowell, Mass.) in 20 mM TRIS-HCl buffer, pH 8, containing 150 mM NaCl and 5 mM DDT in a total volume of 80 μL. Test compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions were also prepared in DMSO, as 40× solutions.
- Immobilized Metal Affinity Polarization (IMAP) Assay The MAPK13 blocking activity of test compounds was determined using an immobilized metal affinity polarization (IMAP) assay containing activated MAPK13, FITC-labeled substrate, and test compound. Full-length 6-His-tagged MAPK13 and constitutively active GST-MKK6 were prepared as described below. Activated MAPK13 was generated in 50 mM Hepes, 10 mM MgCl2 and 1 mM DTT, containing 1 uM MAPK13, 2 uM MKK6 and 50 uM ATP for 1 h at 25° C. MKK6 was removed by incubation with glutathione SEPHAROSE 4B beads (GE Healthcare Biosciences). MAPK13 activation was confirmed by Western blot using anti-phospho-p38-MAPK (T180/Y182) antibody (R& D Systems, Minneapolis, Minn.). IMAP assays were performed in 96-well non-treated half-area black plates or 384-well black plates (Corning Inc., Corning, N.Y.) in a final reaction volume of 20 ul using the linear phase of the rate kinetics. Assay reactions contained 0-100 uM test compound, 5-35 nM (EC80) activated MAPK13, 3 uM.
- Inhibition Assay A fusion protein comprising glutathione S transferase (GST) and human IRE-1alpha (GST-IRE-1alpha) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1alpha activity in vitro. Five ul of a reaction mixture comprising IX reaction buffer (5x reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three ul of a 128 ng/ml IRE-1alpha preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound.After spinning the plates at 1200 rpm for 30 seconds, 3 ul of an IRE-1alpha human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2.
- Lanthascreen Kinase Binding Assay mTOR: Binding Assays are based on the binding and displacement of an Alexa Fluor 647-labeled, ATP-competitive kinase inhibitors to the kinase of interest. Invitrogen's Kinase Tracers have been developed to address a wide range of kinase targets and are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site or to an allosteric site altering the conformation of the ATP site.In the Lanthascreen kinase binding assay, the donor (Eu3+-anti-GST (glutathione 5-transferase) antibody) is excited at 340 nm and will transfer its energy to the acceptor (Alexa Fluor 647-labeled ATP-competitive kinase inhibitor=Tracer-314). The emission from the Tracer-314 (Alexa Fluor 647 inhibitor) can be monitored with a filter centered at 665 nm because it is located between the emission peaks of the donor, which is measured at 615/620 nm. The binding of both, the Tracer-314 and Eu3+-anti-GST antibody, to the kinase results in a high degree of FRET from the Eu3+-donor fluorophore to the Alexa-Fluor 647-acceptor fluorophore on the Tracer-314. Binding of an inhibitor to the kinase competes for binding with the tracer, resulting in a loss of FRET.
- Kinase High ATP Assay MKNK1-inhibitory activity at high ATP of compounds of the present invention after their preincubation with MKNK1 was quantified employing the TR-FRET-based MKNK1 high ATP assay as described in the following paragraphs.A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 uL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 uL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 uL assay volume is 2 mM) and substrate (0.1 uM=>final conc. in the 5 uL assay volume is 0.06 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 ug/mL. The reaction was stopped by the addition of 5 uL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
- MKNK1 Kinase High ATP Assay MKNK1-inhibitory activity at high ATP of compounds of the present invention after their preincubation with MKNK1 was quantified employing the TR-FRET-based MKNK1 high ATP assay as described in the following paragraphs.A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (SEQ ID NO: 1) (C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany)For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 mM at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
- AlphaLISA Assay Table 8: Exemplary MEK inhibitor compounds (0, 0.01, 0.1, or 1 μM) were added to a 384-AlphaPlate (PerkinElmer, 6008350) by a digital liquid dispenser (Tecan D300e) based on a total volume of 20 μL reaction mixture. 5 μL 4×MEK1 stock solution was added to wells A1-P20 by multichannel pipettes. The plate was sealed with clear olefin sealing tape (Thermo Scientific, 232701), centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 30 minutes. 5 μL 4×CRAF stock solution at different concentrations or 5 μL or 10 μL AlphaLISA buffer was added by multichannel pipettes. The plate was sealed, centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 60 minutes. 5 μL 4×glutathione acceptor beads were first added to all wells by multichannel pipettes. The ambient light was subsequently adjusted to <100 lux (light meter) before adding 5 μL 4×nickel chelate donor beads to the same wells. From this step on, the plate was covered with a black plate cover, centrifuged at 1000 rpm for 1 minute, and incubated at room temperature for 60 minutes.
- AlphaScreen Assay Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
- CRBN-DDB1/BRD4 Dimerization Assay AlphaScreen technology was used to detect CRBN-DDB1/BRD4 dimerization induced by dBET molecules. In brief, GST-BRD4[49-170] (Sigma Aldrich) and CRBN-DDB1 (6×HIS-tagged) were diluted to 125 nM and 250 nM, respectively, in assay buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 1 mM TCEP, and 0.1% BSA), and 20 uL of protein mixture was added to each well of a 384-well AlphaPlate (PerkinElmer). Compounds were then added at 100 nL per well from DMSO stock plates using a Janus Workstation (PerkinElmer). After 1 hr incubation at room temperature, Nickel Chelate AlphaLISA Acceptor and Glutathione AlphaLISA Donor beads (PerkinElmer) were diluted in assay buffer to a 2× concentration (20 ng/ul) and added at 20 uL per well. Plates were incubated for 1 hr at room temperature prior to luminescence detection on an Envision 2104 plate reader (PerkinElmer). For competition assays, GST-BRD4[49-170] and CRBN-DDB1 were diluted as above in the presence of 111 nM dBET1. Compound addition and subsequent detection was performed as described above.Data were analyzed and plotted using GraphPad PRISM v6.
- Enzymatic Chymase Assay The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 ul of a 50-fold concentrated solution of test substance in DMSO, 24 ul of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 ul of substrate solution (final concentration 10 uM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtitre plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Mainnedorf, Switzerland). One test compound is tested on the same microtitre plate in 10 different concentrations from 30 uM to 1 nM in a double determination. The data are normalized (enzyme reaction without inhibitor=0% inhibition, all assay components without enzyme=100% inhibition) and IC50 values are calculated using in-house software.
- Enzyme Assay Active GST-LRRK2 (1326-2527), GST-LRRK2[G2019S] (1326-2527), GST-LRRK2[A2016T] (1326-2527) and GST-LRRK2[A2016T+G2019S] (1326-2527) enzyme was purified with glutathione sepharose from HEK293 cell lysate 36 h following transient transfection of the appropriate cDNA constructs. Peptide kinase assays, performed in triplicate, were set up in a total volume of 40 μl containing 0.5 μg LRRK2 kinase (which at approximately 10% purity gives a final concentration of 8 nM) in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 20 μM Nictide, 0.1 μM [γ-32P]ATP ( 500 cpm/pmol) and the indicated concentrations of inhibitor dissolved in DMSO. After incubation for 15 min at 30° C., reactions were terminated by spotting 35 μl of the reaction mix onto P81 phosphocellulose paper and immersion in 50 mM phosphoric acid. Samples were washed extensively and the incorporation of [γ-32P]ATP into Nictide was quantified by Cerenkov counting. IC50 values were calculated with GraphPad Prism using non-linear regression analysis.
- In Vitro hLTC4 Synthase Enzyme Assay (Test B) In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 methyl ester is converted to LTC4 methyl ester. In order to obtain IC50-values for the compounds, the following procedure was used: 10 mL of 0.6 nM of human recombinant purified LTC4 synthase expressed from Pichia Pastoris in buffer 50 mM tris (pH 7.5), 0.05% BSA 0.03% DD M 100 mM NaCl was preincubated for 30 min with 10 mL of substrate mix containing 6 mM LTA4-methyl ester substrate and 1 mM glutathione in 50 mM Tris pH7.5 0.05% BSA 0.03% DDM 100 mM NaCl together with 0.1 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10^−9,5-10^−5 M as well as with DMSO only. The enzymatic reaction is initiated by addition of 10 L LTC4S enzyme solution to 10 μL substrate solution to the assay plate containing 0.5 mL of compound in DMSO or 0.5 mL DMSO control. The reaction is stopped after 30 min by addition of 40 μL 75% (w/v) acetonitrile in H2O.
- Kinase Assay ALK-5 kinase assay methods have been described in the art (see e.g., Laping et al. (2002) Mol. Pharmacol. 2002; 62: 58-62). The compounds named in the specified Examples were tested as follows for inhibition of ALK-5 autophosphorylation activity and of the ALK-5 phosphorylation of α-Casein.Materials: Buffer. 50 mM HEPES, pH 7.6, with 10 mM NaCl, 10 mM MgCl2, and 1 mM DTT. GST-ALK-5 protein 0.44 mg/ml (roughly 7 μM stock). A 1:350 dilution gives a 20 nM stock, which translates to 2 nM final in assay. Human ALK-5 was expressed in Sf9 insect cells infected with Baculovirus expressing a ALK-5 truncation sequence (amino acids H149-M503), fused at the N-terminus to Glutathione S-transferase GST, in a pFastBac vector (Invitrogen). The cells were disrupted by sonication at 4° C. The lysate was centrifuged at 40,000×g for 45 minutes, and the supernatant applied to a 10 ml column of Glutathione Sepharose 4 Fast Flow (Amersham Bioscienses) equilibrated with 100 mM Tris-HCl pH 7.6 buffer containing 300 mM NaCl, 10% glycerol, 1% NP40, 2 mM dithiothreitol (DTT) and one Protease Inhibitor complete EDTA-free tablet per 50 ml (Roche). The column was washed with 5 column volumes of 50 mM Tris HCl pH 8.0 containing 150 mN NaCl, 10% glycerol, 2 mM DTT and one Protease Inhibitor complete EDTA-free tablet per 100 ml. The column was eluted with wash buffer containing 8 mM reduced glutathione. Fractions were collected and dialyzed overnight in 20 mM Tris HCl pH 8.0 containing 10% glycerol, 150 mM NaCl, 2 mM DTT and 1 mM 4-(2-aminoethyl)-bezenesulfonylfluoride.HCl (AEBSF) (Sigma) at 4° C. α-Casein (Sigma, #C8032) is made up at 2 mM in Buffer (50 mg/ml). Cold ATP contains 10 μM cold ATP (from a 10 mM stock in Buffer). Hot ATP consists of 0.5 μCi/well □-33P-ATP (Amersham, AH9968) in Buffer. Assay Buffer Per 10 ml Buffer 1 ml of 500 mM HEPES (pH 7.6) 20 μl of 5 M NaCl 100 μl of 1 M MgCl2 10 μl of 1 M DTT (dithiothreitol) Assay Method:In a 96 well filter-bottom plate (Millipore, #MSDV N6B 50), 58 μl Assay Buffer is added to reach well. Add 10 μl of Cold ATP mix in Assay Buffer, then 10 μl of a 1:10 dilution of α-Casein stock. Then add 2 μl of compound being tested (DMSO) at a 50× final concentration. Hot ATP mix (10 μl) is added, and the reaction is started with the addition of 10 μl of a 1:350 dilution of the ALK-5 protein (2 nM final) in Assay Buffer with 0.05% BSA (Bovine Serum Albumin). The reaction is mixed for 5 minutes at room temperature, and then continued for 145 minutes at room temperature. The reaction is then stopped with the addition of 100 μl of ice-cold 20% TCA (trichloroacetic acid). The assay is then incubated for at least 1 hour at 4° C., and then the contents of each well are filtered by suction through the filter. The wells are washed three times with 200 μl ice-cold 10% TCA. The plate bottom is blotted before and after removing plastic sub-base, and dried overnight at room temperature. Add 30 μl of scintillation fluid, and count 1 minute per well on a Wallac Tri-Lux scintillation counter.
- AlphaScreen Assay 15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (Perkin Elmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HIS Multilabel Reader using the AlphaScreen protocol from Perkin Elmer.
- AlphaScreen Assay NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
- Amplified Luminescent Proximity Homogeneous Assay (ALPHA) The interactions between test compounds and BRD4 protein containing both bromodomain 1 and bromodomain 2 were measured with human BRD4 protein with N-terminal His tag (BPS Bioscience, San Diego, Calif.) using AlphaScreen® assay at room temperature. A 9 μl reaction mixture in BRD Assay Buffer (BPS Bioscience) containing 25 nM BRD4 and test compounds at various concentrations were incubated for 30 minutes followed by additional 30-minute incubation with 1 μl of 20 M histone H4 peptide (residue 1-21) in the presence of 5% DMSO. Test compounds (see Table 4) were assayed at 10 μM or 31.6 μM for screening purpose, while 8 different concentrations (10 nM-10 μM) were used for IC50 measurements. After the incubation, 20 μl of BRD Detection Buffer (BPS Bioscience) containing 10 μg/ml Glutathione Acceptor beads and 10 μg/ml Streptavidin Donor beads (PerkinElmer, Waltham, Mass.) was added and the mixture was incubated for 50 minutes in darkroom. Binding measurements were taken in duplicate at each concentration using EnSpire® Alpha Multimode Plate Reader Model 2390 (PerkinElmer).
- Biochemical TR-FRET Assay To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.
- Enzymatic Chymase Assay The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 ul of a 50-fold concentrated solution of test substance in DMSO, 24 ul of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 ul of substrate solution (final concentration 10 uM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) were combined in a white 384-hole microtitre plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Minnedorf, Switzerland).One test compound is tested on the same microtitre plate in 10 different concentrations from 30 uM to 1 nM in a double determination. The data are normalized (enzyme reaction without inhibitor=0% inhibition, all assay components without enzyme=100% inhibition) and IC50 values are calculated using in-house software. Compounds in the context of the invention which were tested in this assay inhibited chymase activity with an IC50 of less than 10 uM.
- In Vitro Enzyme Assay A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) was obtained from a 500 ml baculovirus-infected insect cell culture and used to measure IRE-1α activity in vitro. Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water were added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution were added to test wells. Three μl of a 128 ng/ml IRE-1α preparation were added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contained only reaction mixture and test compound.After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5′-CAGUCCGCAGCACUG-3′ (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5′ end and Black Hole Quench
- In Vitro hLTC4 Synthase Enzyme Assay In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 methyl ester is converted to LTC4 methyl ester.In order to obtain IC50-values for the compounds, the following procedure was used:10 mL of 0.6 nM of human recombinant purified LTC4 synthase expressed from Pichia Pastoris in buffer 50 mM tris (pH 7.5), 0.05% BSA 0.03% DD M 100 mM NaCl was preincubated for 30 min with 10 mL of substrate mix containing 6 mM LTA4-methyl ester substrate and 1 mM glutathione in 50 mM Tris pH7.5 0.05% BSA 0.03% DDM 100 mM NaCl together with 0.1 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10−9.5-10−5 M as well as with DMSO only.The enzymatic reaction is initiated by addition of 10 μL LTC4S enzyme solution to 10 μL substrate solution to the assay plate containing 0.5 mL of compound in DMSO or 0.5 mL DMSO control-The reaction is stopped after 30 min by addition of 40 μL 75% (w/v) acetonitrile in H2O.After stopping the reaction the LTC4 methyl ester product was detected by LCMSMS,The detected product concentration, with background subtracted, is plotted versus compound concentration and IC50 is determined as 50% of maximum inhibition.
- Inhibition Assay The inhibition of ALK tyrosine kinase activity can be demonstrated using known methods. For example, in one method, compounds can be tested for their ability to inhibit kinase activity of baculovirus-expressed ALK using a modification of the ELISA protocol reported for trkA. Phosphorylation of the substrate, phopholipase C-gamma (PLC-γ) generated as a fusion protein with glutathione-S-transferase (GST) can be detected with europium-labeled anti-phosphotyrosine antibody and measured by time-resolved fluorescence (TRF). In this assay, 96-well plate is coated with 100 μL/well of 10 μg/mL substrate (phospholipase C-γ in tris-buffered saline (TBS). The assay mixture (total volume=100 μL/well) consisting of 20 nM HEPES (pH 7.2, 1 μMATP (Km level), 5 nM MnCl2, 0.1% BSA, 2.5% DMSO, and various concentrations of test compound is then added to the assay plate. The reaction is initiated by adding the enzyme (30 ng/mL ALK) and is allowed to proceed at 37 degrees C. for 15 minutes. Detection of the phosphorylated product can be performed by adding 100 μL/well of Eu-N1 labeled PT66 antibody (Perkim Elmer # AD0041). Incubation at 37° C. for one hour, followed by addition of 100 μL enhancement solution (for example Wallac #1244-10).
- Inhibition of Auto-Phosphorylation of Recombinant Human NF-kappaB-Inducing Kinase (NIK1/AP3K14) Activity NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 100 (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-KK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
- Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity (AlphaScreen) NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen (alphascreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. The final DMSO concentration was 0.7% (v/v) in assays. The assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, and 0.01% Tween 20. The assays were carried out in 384 well Proxiplates (Perkin Elmer). The incubations consisted of the compound, 5 uM Adenosine-5'-triphosphate (ATP), and 1 nM NIK/MAP3K14. Incubations were initiated by the addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 2 h at 25 C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision Multilabel Plate Reader (Perkin Elmer). The signal obtained in the wells was normalized using high (full enzyme activity, 0.7% DMSO) and low controls (no enzyme activity, 0.7% DMSO, no ATP).
- Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
- Potency of IRAK4 Inhibitor Compounds in IRAK4 Enzyme Assay The inhibitory activity of compounds against IRAK4 were determined in an enzymatic assay using mass spectrometry readout. Ten point half-log compound concentration response curves, with a top concentration of 1 μM or 10 μM, were generated from 10 mM stocks of compound solubilized in DMSO using an Echo 655 (Labcyte Inc) and added to 384 well assay plates (Greiner #781280). To the assay plates, 10 μL of human recombinant IRAK4 protein (Life Technologies #PV4002) diluted to a final concentration of 0.2 nM in assay buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl, 5 mM glutathione, 0.01% BSA, 3 mM ATP) was added. The enzyme was incubated with the compounds at room temperature for 15 minutes before a peptide substrate (KKARFSRFAGSSPSQSSMVAR, Innovagen custom synthesis, 10 mM in DMSO) was added to each well to a final concentration of 10 μM using an Echo 655 (Labcyte Inc). After two hours at room temperature, the reaction was stopped with 90 μL of 0.4% formic acid (Merck #33015). The unphosphorylated and phosphorylated peptide were measured by LC-MS/MS on a Waters TQ-S mass spectrometer. Peaks were integrated using the TargetLynx software and the ratios between phosphorylated and unphosphorylated peptides were calculated.
- TR-FRET Based Assay A recombinant tagged FGFR-3 fusion protein [fusion of glutathione-S-transferase (GST) (N-terminally), His6-tag, thrombin cleavage site, and the intracellular part of human FGFR-3 from amino acids R397 to T806 as in NCBI/Protein entry NP_000133.1], expressed in SF9 insect cells using baculovirus expression system and purified via glutathione-S-transferase affinity chromatography, was purchased from Proqinase (product no. 1068-0000-1) and used as enzyme. As substrate for the kinase reaction, the biotinylated peptide biotin-Ahx-AAEEEYFFLFAKKK (C-terminus in amide form) was used which can be purchased, e.g., from Biosyntan (Berlin-Buch, Germany). Usually, test compounds were tested on the same microtiter plate at 11 different concentrations in the range of 20 uM to 0.1 nM (e.g. 20 uM, 5.9 uM, 1.7 uM, 0.51 uM, 0.15 uM, 44 nM, 13 nM, 3.8 nM, 1.1 nM, 0.33 nM, and 0.1 nM) in duplicates for each concentration. The dilution series was prepared separately prior to the assay as 100-fold concentrated stock solutions in DMSO; exact concentrations could vary depending on the pipettor used. For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). 2 ul of a solution of the above FGFR-3 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 16.7 uM; final concentration in the 5 ul assay volume=10 uM) and substrate (0.8 uM; final concentration in the 5 ul assay volume=0.5 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of FGFR-3 fusion protein was adjusted depending on the activity of the enzyme lot and was chosen appropriately to have the assay in the linear range (typical concentrations were in the range of 0.03 ug/ml). The reaction was stopped by the addition of 5 ul of a solution of HTRF detection reagents [100 nM streptavidin-XL665 (Cis Biointernational) and 1 nM PT66-Tb-cryptate, a terbium-cryptate labelled anti-phosphotyrosine antibody (Cis Biointernational; PT66-Eu-chelate from Perkin-Elmer may be used instead), in an aqueous EDTA solution (50 mM EDTA, 0.1% (w/v) BSA in 50 mM HEPES/NaOH pH 7.5)].
- AlphaScreen Assay Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (probe molecule JQ1+ with protein/peptide mix) is pipetted.
- Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader.
- Biochemical Assay The biochemical PIKFyve inhibition assays were run by Carna Biosciences according to proprietary methodology based on the Promega ADP-Glo Kinase assay. A full-length human PIKfyve [1-2098(end) amino acids and S696N, L932S, Q995L,T998S, S1033A and Q1 183K of accession number NP_055855.2] was expressed as N-terminal GST-fusion protein (265 kDa) using baculovirus expression system. GST-PIKfyve was purified by using glutathione sepharose chromatography and used in an ADP-Glo Kinase assay (Promega). Reactions were set up by adding the test compound solution, substrate solution, ATP solution and kinase solution, each at 4 final concentrations. Reactions were prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2), mixed, and incubated in black 384 well polystyrene plates for 1 hour at room temperature. ADP-Glo reagent was then added for 40 minutes, followed by kinase detection reagent for an additional 40 minutes. The kinase activity was evaluated by detecting relative light units on a luminescence plate reader. Samples were run in duplicate from 10 μM to 3 nM. Data were analyzed by setting the control wells (+PIKfyve, no compound) to 0% inhibition and the readout value of background (no PIKfyve) set to 100% inhibition, then the % inhibition of each test solution calculated. IC50 values were calculated from concentration vs % inhibition curves by fitting to a four-parameter logistic curve.
- Biochemical Assay The human tankyrase 1 PARP catalytic domain, TNKS1P, was cloned into a pDONR221 vector using the Invitrogen Gateway Technology. This entry clone was then subcloned into the destination vector pDEST20 to obtain the N-terminal Glutathione S-transferase (GST)-tagged fusion protein. GST-TNKS1 P was then expressed in Sf21 cells using the Invitrogen baculovirus expression system (Invitrogen-Bac-to-Bac Baculovirus Expression System, Version D). The protein was purified by a GSTrap column (GE Healthcare). The N-terminal GST-tagged tankyrase 2 protein PARP domain, TNKS2P, was cloned, expressed, and purified in a similar manner. Human PARP1 (Cat. No. 4668-100-01) and activated DNA (Cat. No. 4671-096-06) were purchased from Trevigen, Inc. PARP2 (Cat. No. ALX-201-064-C020) was purchased from Alexis Biochemical.The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP auto-parsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of p-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water).
- Enzyme Inhibition Assay Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132, AstraZeneca R&D Boston), or JAK3 (amino acids 781-1124, AstraZeneca R&D Boston) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween-20, 50 μg/ml BSA, and 10 mM MgCl2. JAK enzyme was expressed as N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed at their approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations.For JAK1, 4 nM of enzyme was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.3 nM enzyme was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.), For JAK3, 0.1 nM enzyme was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NII2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphotylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition.
- Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen (ascreen) format (Perkin Elmer). All compound6 tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. The final DMSO concentration was 0.7% (v/v) in assays. The assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM NasVCO4, 5 mM MgCE, and 0.01 % Tween 20. The assays were carried out in 384 well Proxiplates (Perkin Elmer). The incubations consisted of the compound, 5 pM Adenosine-5'-triphosphate (ATP), and 1 nM NIK/MAP3K14. Incubations were initiated by the addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 2 h at 25 °C and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor bead6 were added before reading using an EnVision Multilabel Plate Reader (Perkin Elmer). The signal obtained in the wells was normalized using high (full enzyme activity, 0.7% DMSO) and low controls (no enzyme activity, 0.7% DMSO, no ATP). IC50 s were determined by fitting a sigmoidal curve to % inhibition of control versus Log compound concentration.
- PIKfyve Biochemical Assay The biochemical PIKFyve inhibition assays were run by Carna Biosciences according to proprietary methodology based on the Promega ADP-Glo Kinase assay. A full-length human PIKFYVE [1-2098(end) amino acids and S696N, L9325, Q995L, T998S, 51033A and Q1183K of the protein having the sequence set forth in NCBI Reference Sequence No. NP_055855.2] was expressed as N-terminal GST-fusion protein (265 kDa) using baculovirus expression system. GST-PIKFYVE was purified by using glutathione sepharose chromatography and used in an ADP-Glo Kinase assay (Promega). Reactions were set up by adding the test compound solution, substrate solution, ATP solution and kinase solution, each at 4 final concentrations. Reactions were prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2), mixed, and incubated in black 384 well polystyrene plates for 1 hour at room temperature. ADPGlo reagent was then added for 40 minutes, followed by kinase detection reagent for an additional 40 minutes. The kinase activity was evaluated by detecting relative light units on a luminescence plate reader. Samples were run in duplicate from 10 μM to 3 nM. Data was analyzed by setting the control wells (+PIKfyve, no compound) to 0% inhibition and the readout value of background (no PIKfyve) set to 100% inhibition, then the % inhibition of each test solution calculated. IC50 values were calculated from concentration vs % inhibition curves by fitting to a four-parameter logistic curve.
- PIKfyve Biochemical Assay The biochemical PIKFyve inhibition assays were run by Carna Biosciences according to proprietary methodology based on the Promega ADP-Glo Kinase assay. A full-length human PIKFYVE [1-2098(end) amino acids and S696N, L932S, Q995L, T998S, S1033A and Q1183K of the protein having the sequence set forth in NCBI Reference Sequence No. NP_055855.2] was expressed as N-terminal GST-fusion protein (265 kDa) using baculovirus expression system. GST-PIKFYVE was purified by using glutathione sepharose chromatography and used in an ADP-Glo Kinase assay (Promega). Reactions were set up by adding the test compound solution, substrate solution, ATP solution and kinase solution, each at 4x final concentrations. Reactions were prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2), mixed, and incubated in black 384 well polystyrene plates for 1 hour at room temperature. ADP-Glo reagent was then added for 40 minutes, followed by kinase detection reagent for an additional 40 minutes. The kinase activity was evaluated by detecting relative light units on a luminescence plate reader. Samples were run in duplicate from 10 uM to 3 nM. Data was analyzed by setting the control wells (+ PIKfyve, no compound) to 0% inhibition and the readout value of background (no PIKfyve) set to 100% inhibition, then the % inhibition of each test solution calculated. IC50 values were calculated from concentration vs % inhibition curves by fitting to a four-parameter logistic curve.
- Protein Kinase Assay Condition B (Thiol-Free Conditions) A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of the protein kinase. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, Mass., USA) in a 50 microliter reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 microliter of assay buffer (standard buffer) 5 microliter of ATP solution (in H2O) 5 microliter of test compound (in 10% DMSO) 20 microliter enzyme/substrate mix. Each assay for the protein kinase contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 microM Na-orthovanadate, 1 mM TCEP, 50 μg/ml PEG20000, ATP (corresponding to the apparent ATP-Km of the kinase, see Table A), [gamma-33P]-ATP (approx. 6×10×E5 cpm per well), with the protein kinase and relevant substrate being used in pre-determined amounts, depending on the kinase in question. For all experiments labeled as Thiol-free , all glutathione was exchanged from protein preparations so as to be removed from the assay and final buffer conditions contained no thiol-containing reagents. In addition, the DTT in the thiol-containing assays is replaced by TCEP in the thiol-free assays and all enzymes and substrates are produced under thiol-free conditions for the thiol-free assays.
- Time Resolved Fluorescence (TRF) Assay The probe binding HDAC11 assay was performed using a time resolved fluorescence (TRF) assay format. Recombinant N-terminal GST tag full-length human HDAC11 was expressed and purified from baculovirus in Sf9 insect cells (SignalChem, # H93-30G-1000). Each assay was performed in 1536 black well microplates (Corning, #3936) in a final volume of 8 L in assay buffer containing 50 mM HEPES (pH 7.5), 50 mM KCl, 50 mM NaCl, 0.5 mM GSH (L-Glutathione reduced, Sigma #G4251), 0.03% BGG (0.22 M filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). 100 nL of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into respective wells of 1536 assay plates for a final test concentration range of 25 M to 1.3 nM respectively. The final concentration in the assay of HDAC11 and probe (a fluorescein labeled HDAC 11 inhibitor) was 2.5 nM and 20 nM respectively. 4 L of 2 probe and 2 anti-GST Terbium (Cisbio, #61GSTXLB) was added to assay plates followed by 4 L of 2 HDAC11. Plates were incubated for 16 hours at room temperature before time resolved fluorescence was read on the Envision (Excitation at 340 nm, and Emission at 485 nm and 535 nm, Perkin Elmer).
- USP7 Assay A (Ubitquin-Rhodamine110 Assay) Each assay was performed in a final volume of 15 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 1 mM GSH (L-Glutathione reduced: Sigma #G4251), 0.03% BGG (0.22 μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). Nanoliter quantities of either an 8-point or 10-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates (Perkin Elmer, ProxiPlate-384 F Plus, #6008269) for a final test concentration range of either 25 μM to 11 nM or 25 μM to 1.3 nM, respectively. The final concentration of the enzyme (USP7, construct USP7 (208-1102) 6*His, Viva Biotech) in the assay was 62.5 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- AKR1C3-Inhibitory Activity Assay The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3; GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)).For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 μl of a solution of NADPH (20 μM→final concentration in 5 μl of assay volume is 10 μM) and Coumberone (0.6 μM→final concentration in 5 μl of assay volume is 0.3 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of typically 90 min. The concentration of the AKR1C3 and the reaction time was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical AKR1C3 concentrations were in the region of 1 nM. The reaction was stopped by addition of 2.5 μl of a stop solution consisting of 3 μM EM-1404 as inhibitor (U.S. Pat. No. 6,541,463) in 50 mM HEPES pH7.5 (3 μM EM-1404→final concentration in 7.5 μl of assay volume is 1 μM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 μM to 73 pM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 10.9 nM, 3.1 nM, 0.9 nM, 0.25 nM and 73 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in duplicates for each concentration, and the IC50 values were calculated using a 4-parameter fit.
- MKNK1 Kinase High ATP Assay A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (SEQ ID NO: 1, C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition).
- ASK1 Inhibitor Effect Determined by ASK1 Enzymatic Assay The purpose of this assay is to determine the effect of ASK1 inhibitors on the production of ADP by ASK1. The recombinant human ASK1 (hASK1) catalytic domain tagged with Glutathione S-transferase is used, and histidine-tagged full-length human MAP kinase kinase 6 (MKK6) and ATP are the substrate and cofactor, respectively.The assay is done using an ADP-Glo Kinase Assay Kit (Promega, Catalog #V9102) according to the manufacturer's protocol with the following modifications. Briefly, hASK1 (0.25 nM) and MKK6 (300 nM) in a buffer (10 mM MOPS pH 7.0; 10 mM Mg-Acetate; 1 mM DTT; 0.025% NP-40; 0.05% BSA; 1.5% glycerol) are incubated with ASK1 inhibitors at varying concentrations ranging from 10.00 uM to 0.17 nM for 15 minutes, followed by incubation with ATP (100 uM) for 30 minutes at room temperature. ADP-Glo Reagent is added to terminate the kinase reaction and deplete the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP. The newly synthesized ATP is measured using a luciferase/luciferin reaction, and the luminescence determined by Envision (PerkinElmer). The luminescence intensities are analyzed by GeneData, and fit to a 4 parameter dose response-inhibitor logistics curve to determine IC50 values, using the effects of 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-4-methyl-N-{6-[4-(propan-2-yl)-4H-1,2,4-triazol-3-yl]pyridin-2-yl}benzamide as a standard and DMSO vehicle for 100% and 0% inhibition, respectively.
- ELISA-Based p53-MDM2 Binding Assay ELISA plates were coated with equivalent amounts of either glutathione S-transferase (GST) protein or GST-MDM2 (1-188) fusion protein and incubated on a microtiter plate shaker overnight at 4 deg C. After washing the wells five times with PBS containing 0.2 M NaCl, the plates were incubated at 37 deg C with blocking solution (PBS containing 5% nonfat dry milk) for 2 hours. After the plates were washed five times, the GST-p53 was added, and the plates were incubated for an additional 30 minutes at 37 deg C. After another washing step, the plates were incubated with the anti-p53 monoclonal pAb421 (Oncogene Science, Inc.) in blocking solution for 1 hour at 37 deg C. The plates were again washed five times and incubated for 1 hour at 37 deg C with a goat anti-mouse IgG alkaline phosphatase conjugate (Promega, Inc.). Excess antibody was removed with 15 washes, the wells were filled with PBS, and the absorbance at 405 nm was read to establish background readings for the wells. After aspiration of the PBS, coupled antibody was incubated with p-nitrophenyl phosphate reagent solution (Sigma-Aldrich, Inc.). Finally, the A405 was measured with a microplate spectrophotometer (Molecular Devices, Inc.). Data analysis was performed using Microsoft Excel 98 (Microsoft Corp.) and DeltaGraph Pro (DeltaPoint, Inc.). Absorbance values were normalized by subtraction of equivalent GST-seeded well values from their GST-mdm2 (1-188)-seeded neighbors. IC50 values were determined by fitting data to a three-parameter sigmoidal dose-response function.
- Enzymatic Assay Human RET kinase cytoplasmic domain (amino acids 658-1114 of accession number NP_000314.1) was expressed as an N-terminal GST-fusion protein using a baculovirus expression system. GST-RET was purified using glutathione sepharose chromatography. The RET kinase enzymatic assay was performed in a total volume of 10 uL with increasing concentrations of RET kinase inhibitor as a singlet in a 384 well format as follows: RET inhibitor compound plates are prepared by adding 100 nL of RET inhibitor at different concentrations to a 384-well plate. 5 μL/well of a 2× enzyme mix (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); 0.1 mg/mL BSA (bovine serum albumin); 1 mM DTT (dithiothreitol); 0.2 nM RET kinase) was added to the 384-well plate and incubated for 30 minutes at 23° C. 5 μL/well of a 2× substrate mix (50 mM HEPES; 1 mM CHAPS; 0.1 mg/mL BSA; 20 μM adenosine triphosphate; 20 mM MgCl2 and 1 biotinylated peptide substrate) was added and incubated for 1 hour at 23° C. 10 μL/well of 2× stop/detection mix (50 mM HEPES; 0.1% BSA; 800 mM Potassium Fluoride; 50 mM EDTA (Ethylenediaminetetraacetic acid); 200× dilution of Europium Cryptate labeled anti-phosphotyrosine antibody; 62.5 nM Streptavidin-XL665) incubated for 1 hour at 23° C. and read on a Homogenous Time-Resolved Fluorescence reader.
- Enzyme Inhibition Assay Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1)). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2)). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.).
- Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-5-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography. Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
- Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
- Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+ compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
- Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 0.2 nM GST-hMDM2 (aa 1-188), 0.5 nM biotinylated-p53 (aa 1-83), 0.18 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 100 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-5-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography. Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
- ITK Enzyme Assay 1.0 M HEPES Buffer pH 7.5 solution was prepared as follows: 238.3 g HEPES free acid (Sigma) and 800 mL of water were combined, and the mixture was stirred until complete dissolution. The pH was adjusted to 7.5 via titration with 5N NaOH and the volume adjusted to 1000 mL. The solution was filtered and sterilized.ITK assay buffer was prepared as follows: 50 mL of HPLC-grade water was treated with 2 mL of 1.0 M HEPES Buffer, 500 μL of 2% Gelatin (Sigma), 1.0 mL of aqueous MgCl2 solution (1.0 M), and 1.0 mL of aqueous glutathione solution (0.5 M), and the solution was mixed. The solution was brought to 99 mL in a graduated cylinder by addition of water and sterilized through a 0.2 μm filter. 0.1 mL of Brij-35 Surfact-Amps Detergent Solution (10% w/v aqueous solution, ThermoFisher) and 1.0 mL of ATP (Teknova,100 mM) were added and the solution was mixed.Preparation of 1.33 ITK enzyme solution was as follows: 49.99 mL of ITK assay buffer was treated with 4.1 μL of ITK enzyme (ITK FL (N-Flag and C-His tagged, −72 kDa) Lake Pharma, 0.25 mg/ml in a buffer containing 25 mM Tris pH 7.8, 150 mM NaCl, 10% glycerol and 2 mM TCEP) and the mixture was gently agitated. The resulting solution was stored on ice. 30 Minutes prior to use, the enzyme solution was removed from ice and equilibrated to RT by incubation in a RT water bath.
- In Vitro Assay A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) was obtained from a 500 ml baculovirus-infected insect cell culture and used to measure IRE-1α activity in vitro.Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water were added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution were added to test wells. Three μl of a 128 ng/ml IRE-1α preparation were added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contained only reaction mixture and test compound.After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5′-CAGUCCGCAGCACUG-3′ (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5′ end and Black Hole Quencher 2 (BH2) at the 3′ end, were added to each well of a control plate. The plates were again spun at 1200 rpm for 30 seconds. Final concentrations for the assay were: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 μM test compound.
- Inhibition AlphaScreen Assay This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations.
- Kinase Enzymatic Assay Human RET kinase cytoplasmic domain (amino acids 658-1114 of accession number NP_000314.1) was expressed as an N-terminal GST-fusion protein using a baculovirus expression system. GST-RET was purified using glutathione sepharose chromatography. The RET kinase enzymatic assay was performed in a total volume of 10 uL with increasing concentrations of RET kinase inhibitor as a singlet in a 384 well format as follows: RET inhibitor compound plates are prepared by adding 100 nL of RET inhibitor at different concentrations to a 384-well plate. 5 μL/well of a 2× enzyme mix (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); 0.1 mg/mL BSA (bovine serum albumin); 1 mM DTT (dithiothreitol); 0.2 nM RET kinase) was added to the 384-well plate and incubated for 30 minutes at 23° C. 5 μL/well of a 2× substrate mix (50 mM HEPES; 1 mM CHAPS; 0.1 mg/mL BSA; 20 μM adenosine triphosphate; 20 mM MgCl2 and 1 μM biotinylated peptide substrate) was added and incubated for 1 hour at 23° C. 10 μL/well of 2× stop/detection mix (50 mM HEPES; 0.1% BSA; 800 mM Potassium Fluoride; 50 mM EDTA (Ethylenediaminetetraacetic acid); 200× dilution of Europium Cryptate labeled anti-phosphotyrosine antibody; 62.5 nM Streptavidin-XL665) incubated for 1 hour at 23° C. and read on a Homogenous Time-Resolved Fluorescence reader.
- Mdm2-p53 Inhibition AlphaScreen Assay This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations.
- Raf IC50 Assay Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
- Scintillation Proximity Assay (IC50) and PPAR alpha Transactivation Assay (EC50) Human PPAR alpha receptor was expressed as recombinant glutathione-S-transferase (GST)-fusion proteins in Escherichia coli. The purified GST-hPPAR alpha receptor was used in scintillation proximity assay (SPA)-based receptor-binding assays. Briefly, GST-PPAR receptor was combined in SPA buffer with anti-GST antibodies (AmershamBiosciences, Piscataway, NJ), and a radioligand in assay plates. Yttrium silicate protein A-coated SPA beads (Amersham Biosciences) were added. The assay plates in the presence of varying concentrations of test compounds were incubated with shaking at 15 deg C for approximately 16 h. The plates were then counted in a TopCount scintillation counter (Packard Bioscience, Meriden, CT) to determine the displacement of the radioligand from the receptor by the compound. Nonspecific binding was determined by using a 100-fold excess of the respective unlabeled ligand. The results are expressed as IC50 calculated by a four-parameter logistic equation. EC50 values were determined using transactivation assay. COS-1 cells were cultured, and cotransfected with pcDNA3-PPAR/GAL4 expression vector, pUAS(5X)-tk-luc reporter vector, and pCMV-lacZ as an internal control for transactivation efficiency using Lipofectamine (Invitrogen). Varying concentrations of test compounds were incubated with the transfected cells at 37 deg C for 48 h. Cell lysates were then produced with reporter lysis buffer (Promega, Milwaukee, WI), and luciferase activity in cell extracts was determined by using luciferase assay buffer (Promega) in an ML3000 luminometer (Dynatech Laboratories, Chantilly, VA).
- TR-FRET Assay In this assay, a GST-tagged human RORγ ligand binding domain (GST-hRORγ-LBD) interacts with a synthetic biotinylated TRAP220 cofactor peptide containing an LXXLL motif (amino acids 631-655 from NP_004765). The ligand-binding domain (LBD) of RORγ is expressed as fusion protein with GST in BL-21 (BL3) cells using the vector pDEST15. Cells are lysed by lysozyme-treatment and sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. The strong constitutive interaction is disrupted upon binding of functional antagonists. The strength of the interaction is monitored by TR-FRET between streptavidine APC interacting with the biotinylated peptide and Europium-labelled Anti-GST. 6 μl RORγ-LBD-GST solution (final 8.7 nM) in assay buffer (20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl, 0.1% delipidated BSA, 1 mM DTT) are dispensed into black 384 well small volume plates (Greiner #784076). 6 μl TRAP220 peptide (final 400 nM), SA-APC (final 1.6 ng/μl), Eu-anti-GST (final 0.125 ng/μl) in assay buffer and 2 μl test compounds (DMSO stocks prediluted in 20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl) are added and the mixture incubated 60 min at RT in the dark before measuring FRET (excitation 337 nm, emission at 615 and 665 nm).
- Time-Resolved Fluorescence Energy Transfer Assay Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 μL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 μM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument.
- USP7 Assay A (Ubitquin-Rhodamine 110 Assay) Each assay was performed in a final volume of 15 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 1 mM GSH (L-Glutathione reduced; Sigma # G4251), 0.03% BGG (0.22 μM filtered, Sigma, # G7516-25G), and 0.01% Triton X-100 (Sigma, # T9284-10L). Nanoliter quantities of either an 8-point or 10-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates (Perkin Elmer, ProxiPlate-384 F Plus, #6008269) for a final test concentration range of either 25 μM to 11 nM or 25 μM to 1.3 nM, respectively. The final concentration of the enzyme (USP7, construct USP7 (208-1102) 6*His, Viva Biotech) in the assay was 62.5 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems # U-555) concentration was 25 nM with [Ub-Rh110]<
- USP7 Assay A (Ubitquin-Rhodamine 110 Assay) Each assay was performed in a final volume of 15 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.03% BGG (0.22 μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). Nanoliter quantities of either an 8-point or 10-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates (Perkin Elmer, ProxiPlate-384 F Plus, #6008269) for a final test concentration range of either 25 μM to 11 nM or 25 μM to 1.3 nM, respectively. The final concentration of the enzyme (USP7, construct USP7 (208-1102) 6*His, Viva Biotech) in the assay was 62.5 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- Ubiquitin-Rhodamine 110 Assay for USP30 Activity The assay was performed in a final volume of 9 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (IM Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 1 mM GSH (L-glutathione reduced, Sigma-Aldrich, G4251-100G), 0.03% BGG (0.22 μM filtered, Sigma, G7516-25G), and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 25 μM to 1.3 nM, top to lowest dose, respectively. Concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of USP30 (human recombinant USP30, Boston Biochem, cat. #E-582) in the assay was 0.2 nM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, UbiQ-126) concentration was 25 nM with [Ub-Rh110]<
- Ubitquin-Rhodamine 110 Assay Each assay was performed in a final volume of 15 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.03% BGG (0.22 μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). Nanoliter quantities of either an 8-point or 10-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates (Perkin Elmer, ProxiPlate-384 F Plus, #6008269) for a final test concentration range of either 25 μM to 11 nM or 25 μM to 1.3 nM, respectively. The final concentration of the enzyme (USP7, construct USP7 (208-1102) 6*His, Viva Biotech) in the assay was 62.5 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- Inhibitory Activity Assay Essentially, the enzyme activity is measured by quantification of the Coumberol from Coumberone (Halim, M., Yee, D. J., and Sames, D., J. AM. CHEM. SOC. 130, 14123-14128 (2008) and Yee, D. J., Balsanek, V., Bauman, D. R., Penning, T. M., and Sames, D., Proc. Natl. Acad. Sci. USA 103, 13304-13309 (2006)). In this test, the increase of the highly fluorescent Coumberol by NADPH (nicotinamide adenine dinucleotide phosphate)-dependent reduction of the non-fluorescent Coumberone by AKR1C3 can be determined.The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3) (GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)). For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.0 ul of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 3 ul of a solution of NADPH (16.7 uM final concentration in 5 ul of assay volume is 10 uM) and Coumberone (0.5 uM final concentration in 5 ul of assay volume is 0.3 uM) in assay buffer, and the resulting mixture was incubated at 22 ° C. for the reaction time of 90 min. The concentration of the AKR1C3 was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the region of 1 nM. The reaction was stopped by addition of 5 ul of a stop solution consisting of the inhibitor EM-1404 [F. Labrie et al. U.S. Pat. No. 6,541,463, 2003] (2 uM final concentration in 5 ul of assay volume is 1 uM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 uM to 96.8 pM (20 uM, 5.9 uM, 1.7 uM, 0.5 uM, 0.15 uM, 44 nM, 12.9 nM, 3.8 nM, 1.1 nM, 0.3 nM and 96.8 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in double for each concentration, and the IC50 values were calculated using a 4-parameter fit.
- Assay for Inhibition of ALK Tyrosine Kinase Activity The inhibition of ALK tyrosine kinase activity can be demonstrated using known methods. For example, in one method, compounds can be tested for their ability to inhibit kinase activity of baculovirus-expressed ALK using a modification of the ELISA protocol reported for trkA in Angeles, T. S. et al., Anal. Biochem. 1996, 236, 49-55, which is incorporated herein by reference. Phosphorylation of the substrate, phopholipase C-gamma (PLC-γ) generated as a fusion protein with glutathione-S-transferase (GST) as reported in rotin, D. et al., EMBO J. 1992, 11, 559-567, which is incorporated by reference, can be detected with europium-labeled anti-phosphotyrosine antibody and measured by time-resolved fluorescence (TRF). In this assay, 96-well plate is coated with 100 μL/well of 10 μg/mL substrate (phospholipase C-γ in tris-buffered saline (TBS). The assay mixture (total volume=100 μL/well) consisting of 20 nM HEPES (pH 7.2, 1 μMATP (Km level), 5 nM MnCl2, 0.1% BSA, 2.5% DMSO, and various concentrations of test compound is then added to the assay plate. The reaction is initiated by adding the enzyme (30 ng/mL ALK) and is allowed to proceed at 37 degrees C. for 15 minutes. Detection of the phosphorylated product can be performed by adding 100 μL/well of Eu N1 labeled PT66 antibody (Perkim Elmer # AD0041). Incubation at 37° C. for one hour, followed by addition of 100 μL enhancement solution (for example Wallac #1244-10). The plate is gently agitated and after thirty minutes, the fluorescence of the resulting solution can be measured (for example using EnVision 2100 multilabel plate reader from Perkin Elmer).
- BRD4-H4 Tetraacetylated Peptide Inhibition AlphaScreen This assay is used to determine whether the compounds inhibit the interaction between the first (BRD4-BD1) or the second (BRD4-BD2) bromodomain of BRD4 and the tetraacetylated histone H4 peptide.Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (probe molecule JQ1+ with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
- Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320).
- Binding Assay Compounds of the present invention were tested for ability to bind to ROR in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORFigure US09796710-20171024-P00001Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORFigure US09796710-20171024-P00001LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORFigure US09796710-20171024-P00001 inverse agonist T0901317 (SigmaAldrich, Cat. # T2320).
- Biochemical Assay A master mix minus Syk enzyme is prepared containing 1X Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2), 0.5 μM Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% glycerol. A master mix plus Syk enzyme is prepared containing 1X Cell Signaling kinase buffer, 0.5 μM PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, 0.25% glycerol and 0.4 ng/well Syk enzyme. Syk enzyme is purchased from Cell Signaling Technologies, expressed in baculovirus and is an N-terminally GST-tagged full length human wildtype Syk (accession number NM-00377).The Syk protein is purified in one step using glutathione-agarose. The purity of the final protein preparation is assessed by SDS-PAGE and Coomassie staining. A solution of 200 μM ATP is prepared in water and adjusted to pH 7.4 with 1N NaOH. A quantity of 1.25 μL of compounds in 5% DMSO is transferred to a 96-well area Costar polystyrene plate.Compounds are tested singly and with an 11-point dose-responsive curve (starting concentration is 10-1 μM; 1:2 dilution). A quantity of 18.75 μL of master mix minus enzyme (as a negative control) and master mix plus enzyme is transferred to appropriate wells in 96-well area Costar polystyrene plate. 5 μL of 200 μM ATP is added to that mixture in the 96-well area Costar polystyrene plate for final ATP concentration of 40 μM.
- Biochemical binding TR-FRET assay The assay was constructed such that GST tagged Mcl-1 protein, was incubated with a Europium-labeled anti-GST antibody and a HyLite Fluor 647-labeled peptide corresponding to the BH3 domain of BIM. Compound IC50 values were assessed following a 10-point, half-log10 dilution schema starting at 100 μM or 10 μM compound concentration. Specifically, human Mcl-1 enzyme from Mcl-1 (E171-G327) was cloned into an overexpression vector, expressed as an N-terminal GST-tagged fusion protein in E. coli and subsequently purified via Glutathione Sepharose-affinity and size-exclusion chromatography. The assay was performed in 384-Well LV plates (Greiner cat #784075) and run in the presence and absence of the compound of interest. Each well of 12 μL assay mixture contained 10 mM Tris (pH 7.4), 1.0 mM DTT, 0.005% Tween-20, 150 mM NaCl, 10% DMSO, and 1.5 nM GST Mcl-1, 0.5 nM LanthaScreen Eu tagged GST antibody (Invitrogen Catalog # PV5594), 4.0 nM HyLite Fluor 647-labeled BIM peptide [C(Hilyte647 C2 Maleimide)-WIAQELRRIGDEFN (SEQ ID NO:1)]. Reactions were incubated at 24° C. for 90 min before reading on a Tecan M1000 spectrfluorometer with excitation at 340 nm and emission at 612 nm & 665 nm. Subsequently, ratio of fluorescent emission intensity at 665 nm to 612 nm was calculated for each reaction, and the dose-response of the ratio to testing compound concentration was fitted to a select fit model that will provide the best fit quality using automatic parameter to derive IC50 values for each testing compound.
- Biochemical binding TR-FRET assay The assay was constructed such that GST tagged Mcl-1 protein, was incubated with a Europium-labeled anti-GST antibody and a HyLite Fluor 647-labeled peptide corresponding to the BH3 domain of BIM. Compound IC50 values were assessed following a 10-point, half-log10 dilution schema starting at 100 μM or 10 μM compound concentration. Specifically, human Mcl-1 enzyme from Mcl-1 (E171-G327) was cloned into an overexpression vector, expressed as an N-terminal GST-tagged fusion protein in E. coli and subsequently purified via Glutathione Sepharose-affinity and size-exclusion chromatography. The assay was performed in 384-Well LV plates (Greiner cat #784075) and run in the presence and absence of the compound of interest. Each well of 12 μL assay mixture contained 10 mM Tris (pH 7.4), 1.0 mM DTT, 0.005% Tween-20, 150 mM NaCl, 10% DMSO, and 1.5 nM GST Mcl-1, 0.5 nM LanthaScreen Eu tagged GST antibody (Invitrogen Catalog #PV5594), 4.0 nM HyLite Fluor 647-labeled BIM peptide [C(Hilyte647 C2 Maleimide)-WIAQELRRIGDEFN (SEQ ID NO:1)]. Reactions were incubated at 24° C. for 90 min before reading on a Tecan M1000 spectrfluorometer with excitation at 340 nm and emission at 612 nm & 665 nm. Subsequently, ratio of fluorescent emission intensity at 665 nm to 612 nm was calculated for each reaction, and the dose-response of the ratio to testing compound concentration was fitted to a select fit model that will provide the best fit quality using automatic parameter to derive IC50 values for each testing compound. Table 9 provides the results from the TR-FRET Mcl1 binding assay.
- Biological Assay The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.
- Biological Assays for Inhibition of Rho-Associated Protein Kinase Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
- Enzyme Inhibition Assay Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition.
- Evaluation of BRD4 binding inhibition ability The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
- HPK1 Kinase Assay (Biochemical Assay) A recombinant fusion protein consisting of full-length human HPK1 (MAP4K1) with an N-terminal Glutatione S-transferase (GST) tag was produced in insect cells Sf21 using the baculovirus expression system. GST-HPK1 protein was purified from cell lysates by glutathione Sepharose affinity chromatography. The assay is run in three continuous steps: 1) the HPK1 enzymatic kinase reaction, 2) an ATP depletion, and 3) the ADP detection, the steps 2 and 3 are performed with ADP-Glo Kinase Assay kit from Promega (V9101). Test compounds were prepared by 10-point serial dilution in dimethyl sulfoxide (DMSO) and 100 nL of each dilution was spotted onto a 384-well Optiplate (Perkin Elmer and Cat #6007299) by Labcyte Echo. 5 μL kinase reaction buffer (0.02% Brij-35, 2 mM DTT, 50 mM HEPES pH 7.5, MgCl2 10 mM, BSA 0.01% and O-glycerophosphate 12.5 mM) containing HPK1 (3.2 nM) enzyme was transferred to each well and incubated for 15 minutes at room temperature at 60% humidity. The enzymatic reaction was started by adding 5 μl of Start-Mix (10 μM ATP and 3.235 μM MBP). After 120 minutes, the reaction was stopped by adding 5 μL of ADP-Glo reagent (Promega, V9101) and incubating for 40 min. in the dark at 23 C. To determine the level of ADP, 10 μl ADP-Glo Detection solution was added and incubated for 1 hour at 23 C. in the dark. The plate was transferred to a Perkin Elmer EnVision (2104 Multilabel Reader) for luminescence detection and percent inhibition activity and IC50 value were determined using Genedata Screener.
- HTS Assay The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- IRE-1alpha Assay A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1α activity in vitro. Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three μl of a 128 ng/ml IRE-1α preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound. After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2 (BH2) at the 3' end, are added to each well of a control plate. The plates are again spun at 1200 rpm for 30 seconds. Final concentrations for the assay are: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 μM test compound. The plates are covered with lids and incubated for one hour at 30° C. The plates are then transferred to an ACQUEST™ microplate reader. Data is analyzed using data analysis software, and the percent activity of IRE-1α is calculated.
- Inhibition Assay A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1α activity in vitro.Five ul of a reaction mixture comprising 1x reaction buffer (5x reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three ul of a 128 ng/ml IRE-1α preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound.After spinning the plates at 1200 rpm for 30 seconds, 3 ul of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2 (BH2) at the 3' end, are added to each well of a control plate. The plates are again spun at 1200 rpm for 30 seconds. Final concentrations for the assay are: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 uM test compound. The plates are covered with lids and incubated for one hour at 30° C. The plates are then transferred to an ACQUEST microplate reader. Data is analyzed using data analysis software, and the percent activity of IRE-1α is calculated.
- Inhibition Assay Active PLK4 was purified from an E. coli expression system as an amino terminal GST fusion of residues 1-391 of human PLK4. The protein was purified from clarified cell extracts after induction at 15° C. overnight using glutathione sepharose, gel permeation chromatography, and ion exchange (Resource Q). The resulting protein was dephosphorylated with lambda phosphatase (NEB cat #P0753), and resolved from the phosphatase using gluthione sepharose. The dephosphorylated GST-PLK4 was stored in aliquots at −80° C. until use.PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat #A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat #03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat #30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-Cl pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat #9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat #T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
- Inhibition of ALK Tyrosine Kinase Activity The inhibition of ALK tyrosine kinase activity can be demonstrated using known methods. For example, in one method, compounds can be tested for their ability to inhibit kinase activity of baculovirus-expressed ALK using a modification of the ELISA protocol reported for trkA in Angeles, T. S. et al., Anal. Biochem. 1996, 236, 49-55, which is incorporated herein by reference. Phosphorylation of the substrate, phopholipase C-gamma (PLC-γ) generated as a fusion protein with glutathione-S-transferase (GST) as reported in rotin, D. et al., EMBO J. 1992, 11, 559-567, which is incorporated by reference, can be detected with europium-labeled anti-phosphotyrosine antibody and measured by time-resolved fluorescence (TRF). In this assay, 96-well plate is coated with 100 μL/well of 10 μg/mL substrate (phospholipase C-γ in tris-buffered saline (TBS). The assay mixture (total volume=100 μL/well) consisting of 20 nM HEPES (pH 7.2, 1 μMATP (Km level), 5 nM MnCl2, 0.1% BSA, 2.5% DMSO, and various concentrations of test compound is then added to the assay plate. The reaction is initiated by adding the enzyme (30 ng/mL ALK) and is allowed to proceed at 37 degrees C. for 15 minutes. Detection of the phosphorylated product can be performed by adding 100 μL/well of Eu N1 labeled PT66 antibody (Perkim Elmer #AD0041). Incubation at 37° C. for one hour, followed by addition of 100 μL enhancement solution (for example Wallac #1244-10). The plate is gently agitated and after thirty minutes, the fluorescence of the resulting solution can be measured (for example using EnVision 2100 multilabel plate reader from Perkin Elmer).
- Kinase Enzymatic Assay Human RET kinase cytoplasmic domain (amino acids 658-1114 of accession number NP_000314.1) was expressed as an N-terminal GST-fusion protein using a baculovirus expression system. GST-RET was purified using glutathione sepharose chromatography. The RET kinase enzymatic assay was performed in a total volume of 10 uL with increasing concentrations of RET kinase inhibitor as a singlet in a 384 well format as follows: RET inhibitor compound plates are prepared by adding 100 nL of RET inhibitor at different concentrations to a 384-well plate. 5 μL/well of a 2× enzyme mix (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); 0.1 mg/mL BSA (bovine serum albumin); 1 mM DTT (dithiothreitol); 0.2 nM RET kinase) was added to the 384-well plate and incubated for 30 minutes at 23° C. 5 μL/well of a 2× substrate mix (50 mM HEPES; 1 mM CHAPS; 0.1 mg/mL BSA; 20 μM adenosine triphosphate; 20 mM MgCl2 and 1 μM biotinylated peptide substrate) was added and incubated for 1 hour at 23° C. 10 μL/well of 2× stop/detection mix (50 mM HEPES; 0.1% BSA; 800 mM Potassium Fluoride; 50 mM EDTA (Ethylenediaminetetraacetic acid); 200× dilution of Europium Cryptate labeled anti-phosphotyrosine antibody; 62.5 nM Streptavidin-XL665) incubated for 1 hour at 23° C. and read on a Homogenous Time-Resolved Fluorescence reader. IC50s were fitted using GraphPad Prism to a sigmoidal dose response.
- RET Kinase Enzymatic Assay Human RET kinase cytoplasmic domain (amino acids 658-1114 of accession number NP_000314.1) was expressed as an N-terminal GST-fusion protein using a baculovirus expression system. GST-RET was purified using glutathione sepharose chromatography. The RET kinase enzymatic assay was performed in a total volume of 10 uL with increasing concentrations of RET kinase inhibitor as a singlet in a 384 well format as follows: RET inhibitor compound plates are prepared by adding 100 nL of RET inhibitor at different concentrations to a 384-well plate. 5 μL/well of a 2x enzyme mix (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate); 0.1 mg/mL BSA (bovine serum albumin); 1 mM DTT (dithiothreitol); 0.2 nM RET kinase) was added to the 384-well plate and incubated for 30 minutes at 23° C. 5 μL/well of a 2× substrate mix (50 mM HEPES; 1 mM CHAPS; 0.1 mg/mL BSA; 20 μM adenosine triphosphate; 20 mM MgCl2 and 1 μM biotinylated peptide substrate) was added and incubated for 1 hour at 23° C. 10 μL/well of 2x stop/detection mix (50 mM HEPES; 0.1% BSA; 800 mM Potassium Fluoride; 50 mM EDTA (Ethylenediaminetetraacetic acid); 200× dilution of Europium Cryptate labeled anti-phosphotyrosine antibody; 62.5 nM Streptavidin-XL665) incubated for 1 hour at 23° C. and read on a Homogenous Time-Resolved Fluorescence reader. IC50s were fitted using GraphPad Prism to a sigmoidal dose response.
- RET Kinase Enzymatic Assay Human RET kinase cytoplasmic domain (amino acids 658-1114 of accession number NP_000314.1) was expressed as an N-terminal GST-fusion protein using a baculovirus expression system. GST-RET was purified using glutathione sepharose chromatography. The RET kinase enzymatic assay was performed in a total volume of 10 uL with increasing concentrations of RET kinase inhibitor as a singlet in a 384 well format as follows: RET inhibitor compound plates are prepared by adding 100 nL of RET inhibitor at different concentrations to a 384-well plate. 5 μL/well of a 2× enzyme mix (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); 0.1 mg/mL BSA (bovine serum albumin); 1 mM DTT (dithiothreitol); 0.2 nM RET kinase) was added to the 384-well plate and incubated for 30 minutes at 23° C. 5 μL/well of a 2× substrate mix (50 mM HEPES; 1 mM CHAPS; 0.1 mg/mL BSA; 20 μM adenosine triphosphate; 20 mM MgCl2 and 1 μM biotinylated peptide substrate) was added and incubated for 1 hour at 23° C. 10 μL/well of 2× stop/detection mix (50 mM HEPES; 0.1% BSA; 800 mM Potassium Fluoride; 50 mM EDTA (Ethylenediaminetetraacetic acid); 200× dilution of Europium Cryptate labeled anti-phosphotyrosine antibody; 62.5 nM Streptavidin-XL665) incubated for 1 hour at 23° C. and read on a Homogenous Time-Resolved Fluorescence reader. IC50s were fitted using GraphPad Prism to a sigmoidal dose response.
- RORgamma-Ligand Binding Domain TR-FRET Assay Protocol II HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing recombinant protein were lysed in TBS buffer (25 mM Tris, pH 8.0, 150 mM NaCl) under sonication. The lysate was added to the purified RORγ-LBD in a volume equivalent of 0.75 μL lysate (from 30,000 SF9 cells) per 75 femtomol of RORγ-LBD protein. The resulting mixture was diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 0.01% BSA) to obtain RORγ-LBD protein at a final concentration of 3 nM.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC-conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). The EC50 value for test compounds was calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
- Radio-Ligand Binding Assay Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320).
- Raf IC50 Assay Protocol Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 μL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH 7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01%Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 μM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH 7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
- TR-FRET Assay Protocol II HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing recombinant protein were lysed in TBS buffer (25 mM Tris, pH 8.0, 150 mM NaCl) under sonication. The lysate was added to the purified RORγ-LBD in a volume equivalent of 0.75 μL lysate (from 30,000 SF9 cells) per 75 femtomol of RORγ-LBD protein. The resulting mixture was diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 0.01% BSA) to obtain RORγ-LBD protein at a final concentration of 3 nM.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC-conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). The EC50 value for test compounds was calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm
- High ATP Kinase Assay A recombinant tagged FGFR-1 fusion protein [fusion of glutathione-S-transferase (GST) (N-terminally), His6-tag, thrombin cleavage site, and the intracellular part of human FGFR-1 from amino acids G400 to R800 as in GenBank entry NM_015850], expressed in SF9 insect cells using baculovirus expression system and purified via glutathione-agarose affinity chromatography, was purchased from Proqinase (product no. 0101-0000-1) and used as enzyme. As substrate for the kinase reaction, the biotinylated peptide biotin-Ahx-AAEEEYFFLFAKKK (C-terminus in amide form) was used which can be purchased, e.g., from Biosyntan (Berlin-Buch, Germany). Usually, test compounds were tested on the same microtiter plate at 11 different concentrations in the range of 20 uM to 0.1 nM (e.g. 20 uM, 5.9 uM, 1.7 uM, 0.51 uM, 0.15 uM, 44 nM, 13 nM, 3.8 nM, 1.1 nM, 0.33 nM, and 0.1 nM) in duplicates for each concentration. The dilution series was prepared separately prior to the assay as 100-fold concentrated stock solutions in DMSO; exact concentrations could vary depending on the pipettor used. For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). 2 ul of a solution of the above FGFR-1 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 3.3 mM; final concentration in the 5 ul assay volume=2 mM) and substrate (0.16 uM; final concentration in the 5 ul assay volume=0.1 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 15 min at 22° C. The concentration of FGFR-1 fusion protein was adjusted depending on the activity of the enzyme lot and was chosen appropriately to have the assay in the linear range (typical concentrations were in the range of 0.05 ug/ml). The reaction was stopped by the addition of 5 ul of a solution of HTRF detection reagents [25 nM streptavidin-XL665 (Cis Biointernational) and 1 nM PT66-Eu-chelate, an europium-chelate labelled anti-phosphotyrosine antibody (Perkin-Elmer; PT66-Tb-cryptate from Cis Biointernational may be used instead), in an aqueous EDTA solution (50 mM EDTA, 0.1% (w/v) BSA in 50 mM HEPES/NaOH pH 7.5)]. The resulting mixture was incubated for 1 h at 22° C. to allow formation of the complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently, the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidin-XL665. For this, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader [e.g. Rubystar (BMG Labtechnologies, Offenburg, Germany) or Viewlux (Perkin-Elmer)]. The ratio of the emissions at 665 nm and at 620 nm was taken as the measure for the amount of phosphorylated substrate. Data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition), and IC50 values were calculated by a 4-parameter fit using an in-house software.
- MKNK1 Kinase Assay MKNK1-inhibitory activity of compounds of the present invention was quantified employing the MKNK1 TR-FRET assay as described in the following paragraphs. A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) (SEQ ID: 1) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/mi. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein 56 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.1 nM (20 μM, 5.9 μM, 1.7 μM, 0.51 μM, 0.15 μM, 44 nM, 13 nM, 3.8 nM, 1.1 nM, 0.33 nM and 0.1 nM, the dilution series prepared separately before the assay on the level of the 100 fold concentrated solutions in DMSO by serial 1:3.4 dilutions) in duplicate values for each concentration and IC50 values were calculated by a 4 parameter fit.
- Ubitquin-Rhodamine 110 Assay for USP1 Activity The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2 (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 M filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 M (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- Ubitquin-Rhodamine 110 Assay for USP1 Activity The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (1-785, GG670, 671AA)/UAF1 (1-677)-Flag; Viva) in the assay was 100 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<
- Biological Activity of Examples 1 to 30 The inhibitory effects of Examples 1 to 30 on the activity of Human Arginase 1 and Arginase 2 activity were quantified by measuring the formation of the thiol group from thioarginine using recombinant Arginase 1 or Arginase 2 produced from E. coli. The thiol group was detected with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacts with the thiol to give the mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB2−) at 412 nm.The assays were run in clear 384 well plates (Greiner cat no: 781101). Various concentrations of Examples 1 to 30 in 300 nL DMSO were dispensed to assay plates using an Echo acoustic dispenser immediately followed by plate sealing and centrifugation. Two pre-mixes were prepared from reagents thawed immediately before addition to assay plates. Pre-mix one comprised human Arginase 1 or human Arginase 2, at a final concentration of 5 nM and 0.5 mM DTNB in assay buffer, 45 mM HEPES pH7.5, brij 35, 0.045% (w/v) and 100 μM MnCl2. Pre-mix two comprised freshly thawed 0.5 mM thioarginine in assay buffer. Fifteen microlitres of pre-mix one was dispensed to assay plates containing Examples 1 to 30, centrifuged and incubated for 30 minutes at room temperature prior to adding fifteen microlitres of pre-mix two.Assay plates were centrifuged prior to reading absorbance at 412 nm in a Pherastar multi-mode plate reader to collect data at time point 0 (T0). The plates were incubated at room temperature for 60 min prior to reading again to collect data at time point 1 (T1). Data is derived by subtracting the A412 signal measured at T0 (time point 0) from that measured at T1 (time point 1). The data was transformed to % effect using the equation:Compound % effect=100*[(X−min)/(max−min)], where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
- Binding Assay Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 ag of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy[26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy[26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay) The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag™-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
- Inhibitory Activity on Human ACC1 and the ACC2 Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
- Kinase Assay A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
- MDM2-p53 Inhibition AlphaScreen This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (5% DMSO instead of test compound; with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
- Mdm2-p53 Inhibition AlphaScreen This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (5% DMSO instead of test compound; with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
- Mdm2-p53 Inhibition AlphaScreen This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (PerkinElmer). A mix consisting of nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (5% DMSO instead of test compound; with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
- Radio-Ligand RORγ Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H] cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- The Measurement of Inhibitory Activity on Human ACC1 and the ACC2 Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H].+Intensity of [AcCoA-H] ) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
- MKNK1 Kinase High ATP Assay MKNK1-inhibitory activity at high ATP of compounds of the present invention after their preincubation with MKNK1 was quantified employing the TR-FRET-based MKNK1 high ATP assay as described in the following paragraphs.A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (C-terminus in amide form) (SEQ ID: 1) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.1 nM (e.g. 20 μM, 5.9 μM, 1.7 μM, 0.51 μM, 0.15 μM, 44 nM, 13 nM, 3.8 nM, 1.1 nM, 0.33 nM and 0.1 nM, the dilution series prepared separately before the assay on the level of the 100 fold concentrated solutions in DMSO by serial dilutions, the exact concentrations may vary depending on the pipettor used) in duplicate values for each concentration and IC50 values were calculated by a 4 parameter fit.
- Nedd8 Conjugation Inhibitory Activity A purified NAE (heterodimer of APPBP1 and UBA3) solution was prepared in the following manner. The human APPBP1 gene (NCBI Reference Sequence number: NM_003905) region corresponding to amino acids 1 to 534 of human APPBP1 protein (NCBI Reference Sequence number: NP_003896, full length: 534 amino acids) was inserted in pBacPAK9 (produced by Clontech) to construct a plasmid pBacPAK9-APPBP1 for expressing APPBP1 full-length protein having a His tag and a TEV protease-recognition sequence at the N-terminus. Next, the human UBA3 gene (NCBI Reference Sequence number: NM_003968) region corresponding to amino acids 1 to 463 of human UBA3 protein (NCBI Reference Sequence number: NP_003959, full length: 463 amino acids) was inserted in pBacPAK9 to construct a plasmid pBacPAK9-UBA3 for expressing UBA3 full-length protein. The pBacPAK9-APPBP1 or pBacPAK9-UBA3, and BacPAK6 DNA were cotransfected into insect cells (Sf9, produced by Clontech) to produce a recombinant baculovirus containing APPBP1 or UBA3 gene. The APPBP1 gene recombinant baculovirus was mixed with the UBA3 gene recombinant baculovirus, and the resulting mixture was used to infect Sf9 cells. The baculovirus-infected Sf9 cells were incubated at 28° C. with shaking for 72 hours in Grace's Insect Medium (produced by Gibco), and the collected cells were suspended in a lysis buffer (50 mM Tris-HCl, 200 mM NaCl, and 10% glycerol (pH 7.4)), followed by sonication. The sonicated cell solution was centrifuged (40,000×g, for 30 minutes) to obtain the supernatant as a crude extract. The crude extract was fractionated on a HisTrap HP column (produced by GE Healthcare) and a TALON Superflow column (produced by Clontech), followed by the addition of a TEV protease. Then, a His-tag cleavage reaction was performed at 4° C. overnight. The resulting solution was subjected to TALON Superflow column chromatography, and the unadsorbed fraction was collected. This fraction was applied to a HiLoad 16/60 Superdex 75 prep grade column equilibrated with 50 mM Tris-HCl, 200 mM NaCl, and 10% glycerol (pH 7.4), and fractionated. A fraction containing an APPBP1/UBA3 complex was concentrated to obtain a purified NAE solution. The purification above was performed entirely at 4° C. The purified NAE solution was stored at −80° C. until use.A purified GST-UBC12 solution was prepared in the following manner. The human UBC12 gene (NCBI Reference Sequence number: NM_003969) region corresponding to amino acids 1 to 183 of human UBC12 protein (NCBI Reference Sequence number: NP_003960, full length: 183 amino acids) was inserted in pGEX-4T-2 (produced by GE Healthcare) to construct a plasmid pGEX-UBC12 for expressing UBC12 full-length protein having a GST tag at the N-terminus. The pGEX-UBC12 was introduced into Escherichia coli (BL21 (DE3), produced by Stratagene), followed by culture at 37° C. for 2 hours in the presence of 1 mM isopropyl-beta-D-thiogalactopyranoside (produced by Sigma-Aldrich). The collected Escherichia coli was suspended in PBS, followed by sonication. The sonicated cell solution was centrifuged (40,000×g, for 5 minutes) to obtain the supernatant as a crude extract. A Glutathione Sepharose 4B carrier (produced by GE Healthcare) was added to the crude extract, and eluted with 50 mM Tris-HCl (pH 7.9), 150 mM NaCl, and a 10 mM reduced glutathione solution, followed by dialysis with 50 mM HEPES (pH 7.5) and a 0.05% BSA solution to obtain a purified GST-UBC12 solution. The purified GST-UBC12 solution was divided and stored at −80° C. until use.The Nedd8 conjugation inhibitory activity was measured using an AlphaScreen assay system. Each of the purified NAE solution and GST-UBC12 solution was diluted with an assay buffer (50 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.05% BSA) and added to a 384-well plate (#3673, produced by Corning) containing the test compound. After reaction at room temperature for 30 minutes, a solution obtained by diluting ATP and Biotin-Nedd8
- Binding Assay Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H] cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion ( 6×His disclosed as SEQ ID NO: 2). The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H] cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and 1050 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- Biochemical Assay of FASN The biochemical FASN activity testing was performed as 384 well two-time-point kinetic fluorescence intensity assay format in Greiner low volume medium binding 384-well black microtiter plates in a total assay volume of 8 μl and was used for high throughput screen. In each well 3 μl 40 nM human recombinant full-length fatty acid synthase (produced in-house in SF9 cells) were dispensed in the following assay buffer: 50 mM potassium phosphate buffer pH 7.0, 0.005% (w/v) BSA, 2 mM Glutathione, 0.02% Tween-20. Then 2 μl of 200 μM NADPH in assay buffer were added, followed by the addition of the test compounds in 10 dilution concentrations starting with 30 μM (final concentration) to get a final DMSO content of 1% (v/v). The mixture was incubated for at least 15 min at room temperature. After the pre-incubation the enzymatic reaction was started by the addition of 2 μl substrate solution (80 μM acetyl-CoA, 240 μM malonyl-CoA). A first fluorescence intensity measurement (time point one) was performed with an Envision multimode reader (Perkin Elmer LAS Germany GmbH) at excitation wavelength 340 nm (lamp mode) and emission wavelength 460 nm. The reaction was incubated for 30 minutes at room temperature. After this the fluorescence intensity was measured again in the Envision using the same parameters as described above (second time point measurement). The data were analysed by subtracting the first time point measurement value from the second time point measurement value (after the enzymatic reaction). The differences of the emission signals were determined. These reflect directly the conversion rate of NADPH. The full value used was the inhibitor-free reaction. A pharmacological zero value was used like GSK837149A (Sigma-Aldrich) in a final concentration of 5-10 μM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer or Condosseo from GeneData.
- Enzyme Inhibition Studies Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
- Homogenous Time-Resolved Fluorescence Assay The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag -TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.As the potencies of the HDM2 inhibitors increased, an improved HTRF assay (HTRF2 assay) was developed. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
- Inhibitory Activity Against IDH1R132H and IDH1R132C Enzymes The IDH1R132H protein and the IDH1R132C protein were prepared as follows: the IDH1R132H or IDH1R132C gene was integrated into a pET24b(+) vector (Novagen) to prepare a construct for C-terminal fusion of 6× histidine tag. After transformation of Rosetta 2 (DE3) E. coli, the expression of the protein was induced with IPTG. The E. coli was collected and homogenized, followed by the affinity purification of the 6× histidine fusion protein using HisTrap HP columns (GE Healthcare Japan Corp.) and gel filtration purification using Superdex 200 columns (GE Healthcare Japan Corp.) to obtain the IDH1R132H or IDH1R132C protein of interest.IDH1R132H and IDH1R132C each convert 2-oxoglutarate and NADPH to D-2-hydroxyglutarate (2-HG) and NADP+. Therefore, the activity of the IDH1R132H and IDH1R132C enzymes can be measured by detecting NADPH levels.The enzyme inhibitory activity evaluation was carried out as follows: 40 μL each of reaction solutions containing different concentrations of each compound (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 0.5 mg/mL bovine serum albumin, 1 mM reduced glutathione, 40 μM NADPH, 0.5 mM 2-oxoglutarate, 0.5% dimethyl sulfoxide, 50000 to 0.128 nM compound, and 12 nM IDH1R132H or 10 nM IDH1R132C as the enzyme) was added to each well of 384-well plates (Greiner Bio One International GmbH, #781096) and incubated at room temperature. While NADPH-derived fluorescence was occasionally monitored, the reaction was terminated by the addition of 5 μL of 0.5 M EDTA before consumption of NADPH. 5 μL of WST-8 reagent (Dojindo Laboratories, #CK04) was further added and mixed therewith. 15 minutes later, the absorbance at 450 nm was measured using a plate reader (PerkinElmer, Inc., EnVision). The observed absorbance value reflects the amount of residual NADPH. From the absorbance data, the enzyme inhibitory activity of each compound at each concentration was calculated and analyzed using medical statistical analysis software GraphPad Prism to calculate an IC50 value.
- JAK Enzymatic Assay Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
- Radio-Ligand ROR gamma Binding Assay (Assay 1) Compounds of the present invention were tested for ability to bind to ROR gamma in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant ROR gamma Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM ROR gamma LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard ROR gamma inverse agonist T0901317 (SigmaAldrich, Cat. # T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
- Radio-Ligand RORgamma Binding Assay (Assay 1) Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves. Inhibitory constants (Ki) were calculated using the following equation.
- ELISA-Based In Vitro Kinase Assay GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
- ELISA-Based in vitro Kinase Assay GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 uL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad). The concentration of inhibitor showing 50% inhibition as compared with the control was expressed as IC50 (μM).
- Evaluation of ABHD6 Enzyme Inhibitory Activity Assay First, 1-arachidonoyl glycerol (Cayman Chemical) as a substrate was prepared with an assay buffer containing 50 mM tris-HCl (pH 7.4), 100 mM NaCl, and 0.05% BSA, so as to have a final concentration of 10 μmol/L. Then, a compound was added therein so as to have a final concentration of 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, or 10 μmol/L (DMSO: final concentration of 0.3%). In addition, solutions to which DMSO was added so as to have a final concentration of 0.3% were prepared as a group to which the compound was not added. The enzyme reaction was started by adding recombinant human ABHD6 (33-337) prepared with the same assay buffer to a mixed solution of the substrate and the compound so as to have a final concentration of 300 μg/mL. The enzyme reaction was started by adding recombinant human ABHD6 (33-337) prepared with the same assay buffer to a mixed solution of the substrate and the compound so as to have a final concentration of 300 μg/mL. The recombinant human ABHD6 (33-337) was a GST-tagged ABHD6, and one which was expressed in E. coli, then purified with a Glutathione-Sepharose 4B resin, and then concentrated was used. The enzyme reaction was carried out at room temperature using a 384-well microplate made of polypropylene, and wells to which no enzyme was added were designated as a blank group. One hour after the start of the enzyme reaction, methanol containing, as an internal standard substance, arachidonic acid-d8 (Cayman Chemical) and 1% formic acid was added to stop enzymatic reaction. The upper part of the enzyme reaction plate was sealed with aluminum, and centrifugation was performed at 560 g for 5 minutes at room temperature. Then, arachidonic acid as an enzyme reaction product and arachidonic acid-ds as the internal standard substance were quantified with RapidFire (registered trademark)-Mass Spectrometry system. The ratio of the quantitative values of the respective substances was taken, and the inhibition rate of arachidonic acid production at each compound concentration was calculated with the average of the blank group as 100% inhibition and the average of the group to which the compound was not added as 0% inhibition to determine the IC50 value.
- In Vitro Histone Deacetylase Assay I The probe binding HDAC11 assay was performed using a time resolved fluorescence (TRF) assay format. Recombinant N-terminal GST tag full-length human HD AC 11 was expressed and purified from baculovirus in Sf9 insect cells (SignalChem, #H93-30G-1000). Each assay was performed in 1536 black well microplates (Corning, #3936) in a final volume of 8 μL in assay buffer containing 50 mM HEPES (pH 7.5), 50 mM KCl, 50 mM NaCl, 0.5 mM GSH (L-Glutathione reduced, Sigma #G4251), 0.03% BGG (0.22 μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). 100 nL of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into respective wells of 1536 assay plates for a final test concentration range of 25 μM to 1.3 nM respectively. The final concentration in the assay of HD AC 11 and probe (a fluorescein labeled HD AC 11 inhibitor) was 2.5 nM and 20 nM respectively. 4 μL of 2× probe and 2× anti-GST Terbium (Cisbio, #61GSTXLB) was added to assay plates followed by 4 μL of 2×HDAC11. Plates were incubated for 16 hours at room temperature before time resolved fluorescence was read on the Envision (Excitation at 340 nm, and Emission at 485 nm and 535 nm, Perkin Elmer).Data from HD AC 11 Assays were reported as percent inhibition (inh) compared with control wells based on the following equation: % inh=1−((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured time resolved fluorescence. AveLow=average time resolved fluorescence of no enzyme control (n=32). AveHigh=average time resolved fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model205. Data is fitted using the Levenburg Marquardt algorithm.
- Radio-Ligand RORgamma Binding Assay (Assay 1) Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. # NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162x) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 mM and incubated for 10 mM at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. # RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 mM and then for 10 mM without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. # T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves. Inhibitory constants (Ki) were calculated using the following equation, where [RL] is the concentration in the assay and KD is a dissociation constant of 25-hydroxy [26,27-3H]-cholesterol:K i = IC 50 ( 1 + [ RL ] K D ) .
- LATS1 Biochemical Caliper Assay The LATS1 biochemical Caliper assay was performed as following.Human LATS1 kinase domain protein was purchased from Carnabio (catalogue number 01-123; lot 15CBS-0098D). Human LATS1, catalytic domain [589-1130(end) amino acids of accession number NP_004681.1] was co-expressed as N-terminal GST-fusion protein (90 kDa) with human His-tagged MOBKL1A [1-216(end) amino acids of accession number NP_775739.1] using baculovirus expression system. GST-LATS1 was purified by using glutathione sepharose chromatography. The substrate (Fluo-SGKtide; Peptide for LATS1; lot BS-41067) has the following sequence: 5-Fluo-Nva-KKRNRRLSVA-amide (SEQ ID NO: 27) x TFA and was purchased from Biosyntan.The reaction is performed in reaction buffer containing 50 mM Hepes pH 7,5; 0.02% Tween20; 0.02% BSA; 1 mM DTT; 10 uM Na3VO4 and 10 mM beta-Glycerolphosphat and fresh added 1 mM MgCl2 and qsp H2O.The substrate solution (2× conc.) in Reaction Buffer contains 300 μM ATP and 4 μM Fluo-SGKtide.The kinase solution (2× conc.) in Reaction Buffer contains 20 nM LATS1 kinase. 4.5 μL of 2× conc. Kinase solution, 50 nL of 1.8 mM compounds and 4.5 μL of substrate solution were added into 384-well plates black small volume from Greiner and incubated at 32° C. for 1 hour. 15 μL of stop buffer containing 100 mM Hepes pH 7,5; 5% DMSO; 0.1% Coating reagent; 10 mM EDTA and 0.02% Brij35 and qsp H2O to each well to terminate the reaction.Substrates and products were electrophoretically separated using the microfluidic-based Caliper EZ Reader System (Caliper Life Sciences) using a 12 sipper chip (cat 760404). The separation takes place in Coating Buffer (idem Stop buffer) and containing 0.1% Coating reagent CR3 and 0.5% coating reagent CR8 (Perkin Elmer).Plates were read using a LED with an excitation at 488 nm and a detection at 520 nm to 5 quantify the fluorescence intensity. The IC50 is measured when the effect of the compound reduces the product fluorescence signal by 50%.
- Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-XL. The HTS assay was conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). Controls, which contained bead mixture and F-Bim but no test compound, were located in columns 1 and 2 on each plate. Plates were placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C. A glutathione-only bead set control (no associated GST-protein) was incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. Specificity of F-Bim binding was determined with a Positive Control using a block of the F-Bim fluor with a non-fluoresceinated Bim peptide. Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Analysis of the time-resolved acquisition data file uses IDLeQuery software to merge the flow cytometry data files with compound worklist files generated by HyperSip software. In dose response experiments, the assay was performed without compound and with nine different concentrations of compound, from 10 nanoM to 100 microM, to produce a series of 9 data points. IDLeQuery calculates the median channel fluorescence (MCF) for each of these ligand concentrations, generating competition curves. Ligand competition curves were fitted by Prism# software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent (EC50, microM)Compounds with EC50 less than 10 microM and magnitude of response greater than 40% (i.e., Bottom of sigmoidal curve < 0.6 * Top of sigmoidal curve, listed as FIT_PERCENT_SPAN) were said to be "Active".
- PP2C Activity Assay HAB1 and PYL proteins were expressed and purified as described in Park et al. ((2009) Science 324(5930):1068-1071), with minor modifications. To obtain GST-HAB1, the HAB1 cDNA was cloned into pGex-2T. Expression was conducted in BL21[DE3]pLysS host cells. Transformed cells were pre-cultured overnight, transferred to LB medium and cultured at 30° C. to culture A600 of 0.5.The culture was then cooled on ice and MnCl2 added to 4 mM and IPTG added to 0.3 mM. After 16 hours incubation at 15° C., cells were harvested and recombinant proteins were purified on glutathione agarose as described in Park et al. To obtain 6×His-PYL receptor fusion proteins, expression constructs previously described by Okamoto et al. 2013, (PNAS 110(29): 12132-12137) were used. ABA receptors were expressed and purified as described above.For the values shown in Table B5, PP2C activity assays using recombinant receptors and PP2Cs were carried out as follows: purified proteins were pre-incubated in 160 μl assay buffer containing 100 mM Tris-HCl-pH7.9, 100 mM NaCl, 3 μg bovine serum albumin, 0.1% 2-mercaptoethanol, 1 mM MnCl2 with ABA or agonists (compounds of the present invention) for 30 minutes at room temperature. Reactions were started by adding 40 μL of a reaction solution containing 25 mM 4-nitrophenyl phosphate in assay buffer after which absorbance measurements were immediately collected using a 405 nm on Wallac plate reader. Reactions contained 100 nM PP2C and 200 nM PYR/PYL proteins. Some quinabactin analogs possess intrinsic fluorescence, which necessitated the use of the non-fluorescent substrate 4-nitrophenol phosphate for phosphatase assays. For inferring IC50 values, calculations from receptor/PP2C assays containing different concentrations (ranging from 1 μM to 4 nM) were. The obtained dose response data was fit to a log (inhibitor) versus response-(variable slope) model using non-linear regression to infer the IC50s, using Graph Pad Prism 6.0.
- TR-FRET Assay To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 ul lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate. Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.). A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well. The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 us, integration time=200 us). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
- USP7 Assay B (Ubitquin-Rhodamine110 Assay) Each assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2 (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). For follow-up studies, 250 nL of an 8-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates for a final test concentration of 25 μM-11 nM (1.25% DMSO final concentration). Unless otherwise indicated, all follow-up assays were run on triplecate plates. Enzyme (USP7, construct Met (208-1102)-TEV-6*His; Viva Q93009-1) concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was either 75 or 250 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems (biotechne) #U-555) concentration was 25 nM with [Ub-Rh 110]<
- Lanthascreen Kinase Binding Assay for mTOR (G) Binding Assays are based on the binding and displacement of an Alexa Fluor 647-labeled, ATP-competitive kinase inhibitors to the kinase of interest. Invitrogen's Kinase Tracers have been developed to address a wide range of kinase targets and are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site or to an allosteric site altering the conformation of the ATP site.In the Lanthascreen kinase binding assay, the donor (Eu3+-anti-GST (glutathione S-transferase) antibody) is excited at 340 nm and will transfer its energy to the acceptor (Alexa Fluor 647-labeled ATP-competitive kinase inhibitor=Tracer-314). The emission from the Tracer-314 (Alexa Fluor 647 inhibitor) can be monitored with a filter centered at 665 nm because it is located between the emission peaks of the donor, which is measured at 615/620 nm. The binding of both, the Tracer-314 and Eu3+-anti-GST antibody, to the kinase results in a high degree of FRET from the Eu3+-donor fluorophore to the Alexa-Fluor 647-acceptor fluorophore on the Tracer-314. Binding of an inhibitor to the kinase competes for binding with the tracer, resulting in a loss of FRET.50 nL of compound dilutions were dispensed onto white 384-well small volume polystyrene plate. Then 5 ul of GST-mTOR and Europium-anti-GST antibody followed by 5 ul of tracer-314 (final assay volume 10 ul) are incubated at RT. The standard reaction buffer for the Lanthascreen kinase binding assay contained 50 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Plates are read 60 mins later in a Synergy2 reader using an integration time of 0.2 microseconds and a delay of 0.1 microseconds.To calculate the emission ratio, the signal emitted at 665 nm from the acceptor (Alexa Fluor 647-labeled Tracer-314) is divided by the signal emitted at 620 nm from the donor (Eu3+ anti-GST antibody).Control for the 0% inhibition was given by the solvent vehicle of the compounds (90% DMSO in H2O). Control for the relative 100% inhibition was performed by adding 10 uM in the mix containing GST-mTOR and Europium anti-GST antibody. An additional control for the absolute 0% inhibition is given by Eu3+ anti-GST antibody without GST-mTOR. Standard compounds for the lipid kinase panel profiling were used as a reference and included in all assay plates in the form of 8 dilution points.
- Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bfl-1. The multiplex is constructed by using beads for each protein target that have been labeled with varying intensities of red color, so that each assay is built on a unique bead set, and each bead set is associated with a unique optical address. Beads are first washed in buffer for 20 minutes before adding the appropriate GST-Bcl fusion protein. The HTS assay was conducted in 384-well microplates in a total assay volume per well of 10.1 microliters (5 microliters of bead mixture, 0.1 microliters of test compound, and 5 microliters of 100 nM F-Bim in HPSMTB). Test compound concentration was 10 microM. Controls, which contained bead mixture and F-Bim but no test compound, were located in columns 1 and 2 on each plate. Plates were placed horizontal axis on rotators and incubated for 1-2 hours at 4 degrees C. A glutathione-only bead set control (no associated GST-protein) was incorporated into each well as a fluorescence scavenger to determine inherent fluorescent properties (at 530 nm emission) of the test compounds. In the study reported here, the 237 compounds that satisfied the hit selection criterion in the primary screen (change in %Inhibition greater than 40%) were tested in a dose response format to confirm activity and determine potency. Additional compounds, which were actives from other Bcl targets, were also included in the dose response evaluations. Final compound dilutions in DMSO ranged from 1 microM to 10 mM. These dilutions were then diluted 1 to 100 to give an assay concentration range of 10 nanoM to 100 microM. Sample acquisition and preliminary analysis is conducted with the HyperCyt(R) high throughput flow cytometry platform. The HyperCyt system interfaces a flow cytometer and autosampler for high-throughput microliter-volume sampling from 384-well microtiter plates. The stream of particles is excited at 488 nm and 635 nM, and flow cytometric data of light scatter and fluorescence emission at 530 +/- 20 nm (FL1) and emission at 665 +/- 10 nm (FL8) are collected on a Cyan Flow Cytometer (Dako). Compounds with EC50 less than 10 microM and magnitude of response greater than 40% (i.e., Bottom of sigmoidal curve < 0.6 Top of sigmoidal curve, listed as FIT_PERCENT_SPAN) were said to be Active.
- Biological Assay The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
- Biological Assays The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing any recombinant protein were lysed and the lysate was added to the purified RORγ-LBD at 0.25 μl lysate (from 10,000 SF9 cells)/nM purified protein. The mixture was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT) to obtain RORγ-LBD final concentration of 3 nM in 384-well assay plate.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) (SEQ ID NO:2) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
- ITK Enzyme Assay 1.0M HEPES Buffer pH 7.5 solution was prepared as follows: 238.3 g HEPES free acid (Sigma) and 800 mL of water were combined, and the mixture was stirred until complete dissolution. The pH was adjusted to 7.5 via titration with 5N NaOH and the volume adjusted to 1000 mL. The solution was filtered and sterilized. ITK assay buffer was prepared as follows: 50 mL of HPLC-grade water was treated with 2 mL of 1.0M HEPES Buffer, 500 μL of 2% Gelatin (Sigma), 1.0 mL of aqueous MgCl2 solution (1.0M), and 1.0 mL of aqueous glutathione solution (0.5M), and the solution was mixed. The solution was brought to 99 mL in a graduated cylinder by addition of water and sterilized through a 0.2 μm filter. 0.1 mL of Brij-35 Surfact-Amps Detergent Solution (10% w/v aqueous solution, ThermoFisher) and 1.0 mL of ATP (Teknova,100 mM) were added and the solution was mixed. Preparation of 1.33× ITK enzyme solution was as follows: 49.99 mL of ITK assay buffer was treated with 4.1 μL of ITK enzyme (ITK FL (N-Flag and C-His tagged, 72 kDa) Lake Pharma, 0.25 mg/ml in a buffer containing 25 mM Tris pH 7.8, 150 mM NaCl, 10% glycerol and 2 mM TCEP) and the mixture was gently agitated. The resulting solution was stored on ice. 30 Minutes prior to use, the enzyme solution was removed from ice and equilibrated to RT by incubation in a RT water bath. Preparation of 4× ITK substrate solution was as follows: 50 mL of ITK assay buffer was treated with 100 μL of BTK peptide (China Peptide Company, 2 mM stock solution in DMSO). The tube was capped, mixed by gently inverting the tube, and then stored on ice. 30 Minutes prior to use, the substrate solution was removed from ice and equilibrated to RT by incubation in a RT water bath.At the time of assay, 7.5 μL of the 1.33× ITK enzyme solution was added to plate wells containing 0.1 μL of varying concentrations of test compound in DMSO. The plate was incubated 30 min at RT. The plate wells were each treated with 2.5 uL of the 4× ITK substrate solution and the plate was sealed (TopSeal , Perkin Elmer). The plate was spun at 1000 rpm for 30 sec and then incubated for 60 min at RT. The seal was removed, and each well was treated with 10 μL of Stop/Detect Buffer (20 mM HEPES pH 7.5, 0.01% gelatin, 1 nM LANCE PT66 (Perkin Elmer), 16.5 μg/ml Surelight APC (Perkin Elmer), 10 mM EDTA, 250 mM NaCl). The plate was again covered and was spun at 1000 rpm for seconds. The plate was allowed to incubate overnight at RT and in a closed carrier to reduce dehydration.
- USP7 Assay B (Ubitquin-Rhodamine 110 Assay) Each assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2 (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). For follow-up studies, 250 nL of an 8-point, 3-fold serial dilution in DMSO was pre-dispensed into assay plates for a final test concentration of 25 μM-11 nM (1.25% DMSO final concentration). Unless otherwise indicated, all follow-up assays were run on triplecate plates. Enzyme (USP7, construct Met (208-1102)-TEV-6*His; Viva Q93009-1) concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was either 75 pM or 250 pM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems (biotechne)#U-555) concentration was 25 nM with [Ub-Rh110]<
- Human LTC4S Enzymatic Assay LTC4 synthase catalyzes the conversion of Leukotriene A4(LTA4) to Leukotriene C4 (LTC4) in the presence of reduced glutathione (GSH) as a co-substrate. For compound testing, compounds are delivered as 10 mM stock solutions in 90% DMSO in matrix tubes. From this, a 1:3 dilution dose response series is prepared with a starting concentration of 30 μM to 0.1 nM. For the enzymatic assay 97.5 nL of compound/DMSO solution is transferred to each well and 5 μL enzyme solution (assay buffer: 50 mM bis-tris propane pH 7.3, 250 mM NaCl, 10 mM MgCl2, 0.001% MGN3) is added to the wells. The final enzyme concentration in the assay is 0.75 nM. The enzyme compound mixture is incubated at room temperature for 15 minutes prior to the addition of 5 μL substrate solution. As the primary substrate LTA4 is a highly unstable intermediate of the arachidonic acid pathway, LTA4 is substituted for a more stable LTA4 methyl ester form (LTA4-Me) for the purposes of screening. A final substrate concentration of 400 μM GSH and 5 μM LTA4-Me is chosen. The LTA4-Me is obtained commercially in 2% triethylamine/hexane solvent. As this solvent is incompatible with the HTRF assay it has to be exchanged with DMSO according to following procedure: add 50 μL of 100% DMSO to 50 μL LTA4-Me (3 mM) in a 2 mL Eppendorf tube and mix gently by inverting the tube. The triethylamine/hexane is evaporated under a constant argon flux at room temperature. The DMSO-LTA4-Me (3 mM) is aliquoted and stored at −20° C. for not longer than 4 weeks, as it is not stable in DMSO due to its oxidizing properties. Upon addition of the substrate, the plate is immediately placed on a shaker for 5 min at room temperature. Immediately after the 5 min incubation 5 μL of H2O solution is added to all wells to stop the reaction. The plate contents are mixed before the addition of detection reagents. The conversion from LTA4-Me and GSH to LTC4-Me is quantified using a LTC4-Me standard curve ranging from 1.5 μM to 0.08 nM. For the detection of the product of the enzymatic reaction LTC4-Me, the Cisbio LTC4-HTRF kit is used as the assay is compatible with the detection of LTC4-Me. 5 μL of diluted LTC4-d2 conjugate (according to manufacturer's protocol) are added to all wells of the assay plate and the contents gently mixed and incubated for 5 minutes at room temperature. Then 5 μL of the diluted LTC4-Eu3+ cryptate (according to manufacturer's protocol) are added to all wells and the contents of the plate gently mixed and incubated 60 min at room temperature before reading the plate on the Spectramax Paradigm (Molecular Devices) using ratiometric analysis (665/616 nM) and the following setup: number of flashes/well of 30, integration time of 0.3 ms, excitation time of 0.05 ms, positioning delay of 0.03 ms, and a ratio multiplicator of 10000.
- ITK Enzyme Assay 1.0 M HEPES Buffer pH 7.5 solution was prepared as follows: 238.3 g HEPES free acid (Sigma) and 800 mL of water were combined, and the mixture was stirred until complete dissolution. The pH was adjusted to 7.5 via titration with 5N NaOH and the volume adjusted to 1000 mL. The solution was filtered and sterilized.ITK assay buffer was prepared as follows: 50 mL of HPLC-grade water was treated with 2 mL of 1.0 M HEPES Buffer, 500 μL of 2% Gelatin (Sigma), 1.0 mL of aqueous MgCl2 solution (1.0 M), and 1.0 mL of aqueous glutathione solution (0.5 M), and the solution was mixed. The solution was brought to 99 mL in a graduated cylinder by addition of water and sterilized through a 0.2 μm filter. 0.1 mL of Brij-35 Surfact-AmpS Detergent Solution (10% w/v aqueous solution, ThermoFisher) and 1.0 mL of ATP (Teknova, 100 mM) were added and the solution was mixed.Preparation of 1.33×ITK enzyme solution was as follows: 49.99 mL of ITK assay buffer was treated with 4.1 μL of ITK enzyme (ITK FL (N-Flag and C-His tagged, 72 kDa) Lake Pharma, 0.25 mg/ml in a buffer containing 25 mM Tris pH 7.8, 150 mM NaCl, 10% glycerol and 2 mM TCEP) and the mixture was gently agitated. The resulting solution was stored on ice. 30 Minutes prior to use, the enzyme solution was removed from ice and equilibrated to RT by incubation in a RT water bath.Preparation of 4×ITK substrate solution was as follows: 50 mL of ITK assay buffer was treated with 100 μL of BTK peptide (China Peptide Company, 2 mM stock solution in DMSO). The tube was capped, mixed by gently inverting the tube, and then stored on ice. 30 Minutes prior to use, the substrate solution was removed from ice and equilibrated to RT by incubation in a RT water bath.At the time of assay, 7.5 μL of the 1.33×ITK enzyme solution was added to plate wells containing 0.1 μL of varying concentrations of test compound in DMSO. The plate was incubated 30 min at RT. The plate wells were each treated with 2.5 uL of the 4×ITK substrate solution and the plate was sealed (TopSeal , Perkin Elmer). The plate was spun at 1000 rpm for 30 sec and then incubated for 60 min at RT. The seal was removed, and each well was treated with 10 μL of Stop/Detect Buffer (20 mM HEPES pH 7.5, 0.01% gelatin, 1 nM LANCE PT66 (Perkin Elmer), 16.5 μg/ml Surelight APC (Perkin Elmer), 10 mM EDTA, 250 mM NaCl). The plate was again covered and was spun at 1000 rpm for 30 seconds. The plate was allowed to incubate overnight at RT and in a closed carrier to reduce dehydration. The seal was removed, and the fluorescence was read with a plate reader with an excitation wavelength of 665 nm and an emission wavelength of 615 nm.
- ITK Enzyme Assay ITK activity was determined by measuring the effect of a test compound in an ITK enzyme assay. 1.0 M HEPES Buffer pH 7.5 solution was prepared as follows: 238.3 g HEPES free acid (Sigma) and 800 mL of water were combined, and the mixture was stirred until complete dissolution. The pH was adjusted to 7.5 via titration with 5N NaOH and the volume adjusted to 1000 mL. The solution was filtered and sterilized. ITK assay buffer was prepared as follows: 50 mL of HPLC-grade water was treated with 2 mL of 1.0 M HEPES Buffer, 500 μL of 2% Gelatin (Sigma), 1.0 mL of aqueous MgCl2 solution (1.0 M), and 1.0 mL of aqueous glutathione solution (0.5 M), and the solution was mixed. The solution was brought to 99 mL in a graduated cylinder by addition of water and sterilized through a 0.2 μm filter. 0.1 mL of Brij-35™ Surfact-Amps Detergent Solution (10% w/v aqueous solution, ThermoFisher) and 1.0 mL of ATP (Teknova, 100 mM) were added and the solution was mixed. Preparation of 1.33×ITK enzyme solution was as follows: 49.99 mL of ITK assay buffer was treated with 4.1 μL of ITK enzyme (ITK FL (N-Flag and C-His tagged, 72 kDa) Lake Pharma, 0.25 mg/ml in a buffer containing 25 mM Tris pH 7.8, 150 mM NaCl, 10% glycerol and 2 mM TCEP) and the mixture was gently agitated. The resulting solution was stored on ice. 30 Minutes prior to use, the enzyme solution was removed from ice and equilibrated to RT by incubation in a RT water bath. Preparation of 4×ITK substrate solution was as follows: 50 mL of ITK assay buffer was treated with 100 μL of BTK peptide (China Peptide Company, 2 mM stock solution in DMSO). The tube was capped, mixed by gently inverting the tube, and then stored on ice. 30 Minutes prior to use, the substrate solution was removed from ice and equilibrated to RT by incubation in a RT water bath. At the time of assay, 7.5 μL of the 1.33×ITK enzyme solution was added to plate wells containing 0.1 μL of varying concentrations of test compound in DMSO. The plate was incubated 30 min at RT. The plate wells were each treated with 2.5 uL of the 4×ITK substrate solution and the plate was sealed (TopSeal, Perkin Elmer). The plate was spun at 1000 rpm for 30 sec and then incubated for 60 min at RT. The seal was removed, and each well was treated with 10 μL of Stop/Detect Buffer (20 mM HEPES pH 7.5, 0.01% gelatin, 1 nM LANCE PT66 (Perkin Elmer), 16.5 μg/ml Surelight APC (Perkin Elmer), 10 mM EDTA, 250 mM NaCl). The plate was again covered and was spun at 1000 rpm for 30 seconds. The plate was allowed to incubate overnight at RT and in a closed carrier to reduce dehydration. The seal was removed, and the fluorescence was read with a plate reader with an excitation wavelength of 665 nm and an emission wavelength of 615 nm. The concentrations and resulting effect values for the tested compound were plotted and the concentration of compound required for 50% effect (IC50) was determined with the four-parameter logistic dose response equation.
- Protein kinase assay (Condition B: Thiol-free Conditions) The IC50 profile of compounds was determined using one protein kinase in a customized, thiol free assay. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each test compound in singlicate against each kinase of interest. Prior to testing, the 1×10−03 M stock solutions in column 2 of the master plates were subjected to a serial, semi-logarithmic dilution using 100% DMSO as a solvent. This resulted in 10 distinct concentrations, with a dilution endpoint of 3×10−08 M/100% DMSO in column 12. Column 1 and 7 were filled with 100% DMSO as controls. Subsequently, 2×10 microliter from each well of the serial diluted copy plates were aliquoted with a 96 channel pipettor into two identical sets of compound dilution plates . All plates were barcoded for automated identification and tracking purposes. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each compound in singlicate. All compounds were stored as powder until being solubilized in DMSO. Solubilized compounds were stored as 1×10−02 M/100% DMSO stock solutions. Prior to the assay process, 90 microliters of H2O were added to each well of a set of compound dilution plates. To minimize potential precipitation, the H2O was added to each plate only a few minutes before the transfer of the compound solutions into the assay plates. Each plate was shaken thoroughly, resulting in compound dilution plates with a final of 10% DMSO. For each assay, 5 microliters of solution from each well of the compound dilution plates/10% DMSO were transferred into the assay plate. The final volume of the assay was 50 μl. All compounds were tested at 10 final assay concentrations in the range from 1×10−05 M to 3×10−10 M, in singlicate. The final DMSO concentration in the reaction cocktails was 1% in all cases. A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of the protein kinase. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, Mass., USA) in a 50 microliter reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 microliter of assay buffer (standard buffer) 5 microliter of ATP solution (in H2O) 5 microliter of test compound (in 10% DMSO) 20 microliter enzyme/substrate mix. Each assay for the protein kinase contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 microM Na-orthovanadate, 1 mM TCEP, 50 μg/ml PEG20000, ATP (corresponding to the apparent ATP-Km of the kinase, see Table A), [gamma-33P]-ATP (approx. 6×10×E5 cpm per well), with the protein kinase and relevant substrate being used in pre-determined amounts, depending on the kinase in question. For all experiments labeled as Thiol-free , all glutathione was exchanged from protein preparations so as to be removed from the assay and final buffer conditions contained no thiol-containing reagents. This was done so there would be no interference with the key cysteines in the proteins of interest.
- kinetic binding analysis Dual histidine and Avi tagged human EIF4E (His6-3C-avi-eIF4E) was expressed in 8 L of TB Media. Induction by 0.4 mM IPTG occurred at 2.0 OD600, and cells were harvested at 15 OD600. 225 gram pellet was diluted in buffer (50 mM Tris, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, pH 7.5 containing 10% glycerol, protease inhibitors, and DNase) to volume of 600 mL and passed once over the microfluidizer. Sample was run on 5 mL HisTrap HP IMAC at 4.0 mL/min IMAC with a 25 mM to 500 mM imidazole gradient for one column volume. GST-PreScission Protease (2 mg, made in-house) was added to sample and allowed to react overnight at 4° C. The cleaved pool was passed over 0.5 mL GST and 0.5 IMAC resin in a gravity column. Sample volume was increased to 800 mL using 50 mM Tris pH 7.5, 1 mM TCEP and passed over a 5 mL hiTrap SP FF at 4.0 mL/min with a 0 to 1 M NaCl gradient over 20 column volumes. Sample was then injected onto a 124 mL S75 Gel Filtration Column at 20 mg/mL. Final Avi-eIF4E (26931 Da) was diluted to 1 mg/mL in 1× Bicine buffer to a volume of 2.5 mg and mixed with ATP/Biotin Mix (10 mM ATP, 10 mM Mg(OAc)2, 50 μM d-biotin final). Biotin Ligase (25 μg BirA produced in house) was added to reaction. Reactions were performed with mixing (500 rpm) on Eppendorf ThermoMixer R at 30° C. for 60 minutes and checked for completeness using LC-MS. To the sample, 100 μl of immobilized glutathione (1:1 with buffer) was add and mixed for 15 min at 4° C. to bind C3 and Bir3 and removed by centrifugation. The sample was buffer exchanged using two consecutive PD-10 columns equilibrated with 20 mM HEPES, 100 mM KCl, 1 mM DTT, pH 7.5.Due to low eIF4E stability, the streptavidin coated chip was prepared and run at 10° C. on the Biacore T200. The eIF4E (0.04 mg/mL, 150 μl) was bound to the sample channel of Series S Sensor Chip SA (GE Life Sciences, BR-1005-31) to surface density of 5000 to 7000 RU (Response Units). Buffer flowed over the chip at 30 μL/min, using 1×PBS, 50 mM NaCl, 0.1% Glycerol, 0.1% CHAPS, and 1% DMSO. Samples of various cap analogs were diluted to various concentrations in a range of 100 μM to less than 1 nM. Samples were injected into the Biacore chip with a two minute association time and a five minute dissociation time. Several buffer injections were done for each sample for blank subtraction.Analysis was done for all sets using Biacore T200 evaluation software. Binding analysis for all compounds is reported as response units at 1 micromolar compound where a higher value for RU is interpreted as greater ligand binding to the surface immobilized eIF4E protein. A subset of compounds was further characterized to determine dissociation constants using kinetic binding. Steady State Affinity fits were done with default settings (4 seconds before injection stop with 5 second window). Kinetic fits were normally done with 1:1 binding model, with constant RI=0 and all other variables set to fit globally.
- Assay Condition B The IC50 profile of compounds was determined using one protein kinase in a customized, thiol free assay. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each test compound in singlicate against each kinase of interest. Prior to testing, the 1×10−03 M stock solutions in column 2 of the master plates were subjected to a serial, semi-logarithmic dilution using 100% DMSO as a solvent. This resulted in 10 distinct concentrations, with a dilution endpoint of 3×10−08M/100% DMSO in column 12. Column 1 and 7 were filled with 100% DMSO as controls. Subsequently, 2×10 microliter from each well of the serial diluted copy plates were aliquoted with a 96 channel pipettor into two identical sets of compound dilution plates. All plates were barcoded for automated identification and tracking purposes. IC50 values were measured by testing 10 concentrations (1×10−05 M to 3×10−10 M) of each compound in singlicate. All compounds were stored as powder until being solubilized in DMSO. Solubilized compounds were stored as 1×10−02 M/100% DMSO stock solutions. Prior to the assay process, 90 microliters of H2O were added to each well of a set of compound dilution plates. To minimize potential precipitation, the H2O was added to each plate only a few minutes before the transfer of the compound solutions into the assay plates. Each plate was shaken thoroughly, resulting in compound dilution plates with a final of 10% DMSO. For each assay, 5 microliters of solution from each well of the compound dilution plates/10% DMSO were transferred into the assay plate. The final volume of the assay was 50 μl. All compounds were tested at 10 final assay concentrations in the range from 1×10−05 M to 3×10−10 M, in singlicate. The final DMSO concentration in the reaction cocktails was 1% in all cases. A radiometric protein kinase assay (33PanQinase Activity Assay) was used for measuring the kinase activity of the protein kinase. All kinase assays were performed in 96-well FlashPlates from PerkinElmer (Boston, Mass., USA) in a 50 microliter reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 microliter of assay buffer (standard buffer) 5 microliter of ATP solution (in H2O) 5 microliter of test compound (in 10% DMSO) 20 microliter enzyme/substrate mix. Each assay for the protein kinase contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 microM Na-orthovanadate, 1 mM TCEP, 50 μg/ml PEG20000, ATP (corresponding to the apparent ATP-Km of the kinase, see Table A), [gamma-33P]-ATP (approx. 6×10×E5 cpm per well), with the protein kinase and relevant substrate being used in pre-determined amounts, depending on the kinase in question. For all experiments labeled as Thiol-free , all glutathione was exchanged from protein preparations so as to be removed from the assay and final buffer conditions contained no thiol-containing reagents. This was done so there would be no interference with the key cysteines in the proteins of interest.For data analysis, the median value of the counts in column 1 (n=8) of each assay plate was defined as low control. This value reflects unspecific binding of radioactivity to the plate in the absence of a protein kinase but in the presence of the substrate. The median value of the counts in column 7 of each assay plate (n=8) was taken as the high control, i.e. full activity in the absence of any inhibitor. The difference between high and low control was taken as 100% activity. As part of the data evaluation the low control value from a particular plate was subtracted from the high control value as well as from all 80 compound values of the corresponding plate. The residual activity (in %) for each well of a particular plate was calculated by using the following formula:Res.
- RIPK1 HTRF Binding Assay A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.Cloning and Baculovirus Expression of RIPK1 ConstructThe coding region of human RIPK1(1-324) flanked by NdeI site at 5′ end and stop codon TGA and XhoI site at 3′ end was codon optimized and gene synthesized at GenScript USA Inc. (Piscataway, N.J.) and subcloned into a modified pFastBacl vector (Invitrogen, Carlsbad, Calif.) with N-terminal His-GST-TVMV tag, to generate His-GST-TVMV-hRIPK1(1-324)-pFB. The fidelity of the synthetic fragment was confirmed by sequencing.Baculovirus was generated for the construct using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. Briefly, recombinant bacmid was isolated from transformed DH10Bac E. coli competent cells (Invitrogen) and used to transfect Spodoptera frugiperda (Sf9) insect cells (Invitrogen). Baculovirus was harvested 72 hours post transfection and a virus stock was prepared by infecting fresh Sf9 cells at a 1/1000 (v/v) ratio for 66 hours.For large scale protein production, Sf9 cells (Expression System, Davis, Calif.) grown in ESF921 insect medium (Expression System) at 2×106 cells/ml were infected with virus stock at a 1/100 (v/v) ratio for 66 hours. The production was carried out either at a 10 L scale in a 22 L cellbag (GE Healthcare Bioscience, Pittsburgh, Pa.) or at a 20 L scale in a 50 L cellbag using WAVE-Bioreactor System 20/50 (GE Healthcare Bioscience). The infected cells were harvested by centrifugation at 2000 rpm for 20 min at 4° C. in a SORVALL RC12BP centrifuge. The cell pellets was stored at −70° C. before protein was purified.Purification of His-GST-TVMV-hRIPK1(1-324)RIPK1 containing cell paste was resuspended in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole, 5% glycerol, 5 mM MgSO4, 1 mM TCEP, 25 U/ml Benzonase, and Complete Protease Inhibitor tablets (1/50 ml, Roche Diagnostics, Indianapolis, Ind.). The cells were lysed by nitrogen cavitation using an unstirred pressure vessel @ 525 PSI (Parr Instrument Company, Moline, Ill.). The suspension was clarified by centrifugation at 136,000×g for 40 min, at 4° C. The lysate was decanted from the pellet and passed through a 5 ml NiNTA Superflow cartridge (Qiagen, Valencia, Calif.) using an AKTA Pure (GE Healthcare). Column was eluted with 10 CV linear gradient into 50 mM Tris 7.5, 150 mM NaCl, 500 mM imidazole, 5% glycerol, 1 mM TCEP. Peak fractions were pooled and loaded directly onto 5 ml GSTrap 4B column (GE Healthcare). Column was washed with 50 mM Tris 7.0, 150 mM NaCl, 5% glycerol, 1 mM DTT and eluted in 10 CV linear gradient into 50 mM Tris 8.0, 150 mM NaCl, 20 mM reduced glutathione, 5% glycerol, 1 mM DTT. Fractions identified by SDS-PAGE as containing RIPK1 were pooled and concentrated using 30 kDa MWCO spin concentrators (Amicon Ultra-15, Millipore, Billerica, Mass.) and loaded onto a HiLoad 26/600 Superdex 200 column (GE Healthcare) equilibrated in 25 mM Tris 7.5, 150 mM NaCl, 2 mM TCEP, 5% glycerol. The RIPK1 protein eluted as a dimer off the SEC column.The yield was 8 mg/L with a purity >95% as determined by Coomassie staind SDS-PAGE gel analysis.
- RIPK1 HTRF Binding Assay A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPKl (1-324) in FRET Buffer (20 mM HEPES, 10 mM MgC12, 0.015% Brij-35, 4mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.Cloning and Baculovirus Expression of RIPK1 ConstructThe coding region of human RIPKl(l-324) flanked by Ndel site at 5' end and stop codon TGA and Xhol site at 3' end was codon optimized and gene synthesized at GenScript USA Inc. (Piscataway, NJ) and subcloned into a modified pFastBacl vector (Invitrogen, Carlsbad, CA) with N-terminal His-GST-TVMV tag, to generate His-GST-TVMV-hRIPKl (1-324)-pFB. The fidelity of the synthetic fragment was confirmed by sequencing.Baculovirus was generated for the construct using the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. Briefly, recombinant bacmid was isolated from transformed DHIOBac E.coli competent cells (Invitrogen) and used to transfect Spodoptera frugiperda (Sf9) insect cells (Invitrogen). Baculovirus was harvested 72 hours post transfection and a virus stock was prepared by infecting fresh Sf9 cells at a 1/1000 (v/v) ratio for 66 hours.For large scale protein production, Sf9 cells (Expression System, Davis, CA) grown in ESF921 insect medium (Expression System) at 2 x 106 cells/ml were infected with virus stock at a 1/100(v/v) ratio for 66 hours. The production was carried out either at a 10 L scale in a 22 L cellbag (GE Healthcare Bioscience, Pittsburgh, PA) or at a 20 L scale in a 50 L cellbag using WAVE-Bioreactor System 20/50 (GE Healthcare Bioscience). The infected cells were harvested by centrifugation at 2000 rpm for 20 min at 4 °C in a SORVALL RC12BP centrifuge. The cell pellets was stored at -70 °C before protein was purified.Purification of His-GST-TVMV-hRIPKl(l-324)RIPK1 containing cell paste was resuspended in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole, 5% glycerol, 5 mM MgSCE, 1 mM TCEP, 25 U/ml Benzonase, and Complete Protease Inhibitor tablets (1/50 ml, Roche Diagnostics, Indianapolis, IN). The cells were lysed by nitrogen cavitation using an unstirred pressure vessel @ 525 PSI (Parr Instrument Company, Moline, IL). The suspension was clarified by centrifugation at 136,000 x g for 40 min, at 4 °C. The lysate was decanted from the pellet and passed through a 5 ml NiNTA Superflow cartridge (Qiagen, Valencia, CA) using an AKTA Pure (GE Healthcare). Column was eluted with 10 CV linear gradient into 50 mM Tris 7.5, 150 mM NaCl, 500 mM imidazole, 5% glycerol, 1 mM TCEP. Peak fractions were pooled and loaded directly onto 5 ml GSTrap 4B column (GE Healthcare). Column was washed with 50 mM Tris 7.0, 150 mM NaCl, 5% glycerol, 1 mM DTT and eluted in 10 CV linear gradient into 50 mM Tris 8.0, 150 mM NaCl, 20 mM reduced glutathione, 5% glycerol, 1 mM DTT. Fractions identified by SDS-PAGE as containing RIPK1 were pooled and concentrated using 30 kDa MWCO spin concentrators (Amicon Ultra-15, Millipore, Billerica, MA) and loaded onto a HiLoad 26/600 Superdex 200 column (GE Healthcare) equilibrated in 25 mM Tris 7.5, 150 mM NaCl, 2 mM TCEP, 5% glycerol. The RIPK1 protein eluted as a dimer off the SEC column.The yield was ~8 mg/L with a purity >95% as determined by Coomassie stain SDS-PAGE gel analysis.