- Zhou, B; Wang, Y; Zhang, C; Yang, G; Zhang, F; Yu, B; Chai, C; Cao, Z Ribemansides A and B, TRPC6 Inhibitors from Ribes manshuricum That Suppress TGF-β1-Induced Fibrogenesis in HK-2 Cells. J Nat Prod 81: 913-917 (2018)
- Kinsella, T; Gelman, M; Hong, H; Darwish, IS; Singh, R; Yu, J; Borzilleri, RM; Velaparthi, U; Liu, P; Darne, CP; Rahaman, H; Warrier, JS TGF-β inhibitors US Patent US10287295 (2019)
- Kinsella, T; Gelman, M; Hong, H; Darwish, IS; Singh, R; Yu, J; Borzilleri, RM; Velaparthi, U; Liu, P; Dame, CP; Rahaman, H; Warrier, JS TGF-beta inhibitors US Patent US11352360 (2022)
- Schwalbe, T; Huebner, H; Gmeiner, P Development of covalent antagonists for β1- and β2-adrenergic receptors. Bioorg Med Chem 27: 2959-2971 (2019)
- Harikrishnan, LS; Fink, BE; Borzilleri, RM; Tonukunuru, G; Sankara Warrier, J TGF beta receptor antagonists US Patent US10399987 (2019)
- Harikrishnan, LS; Fink, BE; Borzilleri, RM; Tonukunuru, G; Rahaman, H; Warrier, JS; Seshadri, B TGFβ receptor antagonist US Patent US10336761 (2019)
- Kakeya, H; Hattori, A; Takasu, Y; Fujii, N; Oishi, S; Kojima, S; Hara, M TGF-β signal transduction inhibitor US Patent US8772297 (2014)
- TGF-ß INHIBITOR COMPOUND AND USE THEROF
- TGF-B INHIBITOR COMPOUND AND USE THEREOF
- Bonafoux, D; Chuaqui, C; Boriack-Sjodin, PA; Fitch, C; Hankins, G; Josiah, S; Black, C; Hetu, G; Ling, L; Lee, WC 2-Aminoimidazoles inhibitors of TGF-beta receptor 1. Bioorg Med Chem Lett 19: 912-6 (2009)
- Suto, MJ; Gupta, V; Mathew, B; Zhang, W; Pallero, MA; Murphy-Ullrich, JE Identification of Inhibitors of Thrombospondin 1 Activation of TGF-β. ACS Med Chem Lett 11: 1130-1136 (2020)
- Sawyer, JS Substituted imidazoles for the inhibition of TGF-β and methods of treatment US Patent US12077548 (2024)
- Sawyer, JS Tri-substituted imidazoles for the inhibition of TGF beta and methods of treatment US Patent US11124509 (2021)
- Bijian, K; Wernic, D; Nivedha, AK; Su, J; Lim, FPL; Miron, CE; Amzil, H; Moitessier, N; Alaoui-Jamali, MA Novel Aurora A and Protein Kinase C (α, β1, β2, and θ) Multitarget Inhibitors: Impact of Selenium Atoms on the Potency and Selectivity. J Med Chem 65: 3134-3150 (2022)
- Geldenhuys, WJ; Nakamura, H 3D-QSAR and docking studies on transforming growth factor (TGF)-beta receptor 1 antagonists. Bioorg Med Chem Lett 20: 1918-23 (2010)
- BULLOUGH, DA; FOULKES, JG; Beeley, NR; Crystal, R METHODS FOR TREATING A PULMONARY DISEASE WITH AN ALK-5 (TGF BETA R1) INHIBITOR US Patent US20240307363 (2024)
- Callahan, JF; Burgess, JL; Fornwald, JA; Gaster, LM; Harling, JD; Harrington, FP; Heer, J; Kwon, C; Lehr, R; Mathur, A; Olson, BA; Weinstock, J; Laping, NJ Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5). J Med Chem 45: 999-1001 (2002)
- Wang, H; Chen, M; Sang, X; You, X; Wang, Y; Paterson, IC; Hong, W; Yang, X Development of small molecule inhibitors targeting TGF-β ligand and receptor: Structures, mechanism, preclinical studies and clinical usage. Eur J Med Chem 191: (2020)
- Goldberg, FW; Ward, RA; Powell, SJ; Debreczeni, JE; Norman, RA; Roberts, NJ; Dishington, AP; Gingell, HJ; Wickson, KF; Roberts, AL Rapid generation of a high quality lead for transforming growth factor-beta (TGF-beta) type I receptor (ALK5). J Med Chem 52: 7901-5 (2009)
- Schade, D; Lanier, M; Willems, E; Okolotowicz, K; Bushway, P; Wahlquist, C; Gilley, C; Mercola, M; Cashman, JR Synthesis and SAR of b-annulated 1,4-dihydropyridines define cardiomyogenic compounds as novel inhibitors of TGFß signaling. J Med Chem 55: 9946-57 (2012)
- Roth, GJ; Heckel, A; Brandl, T; Grauert, M; Hoerer, S; Kley, JT; Schnapp, G; Baum, P; Mennerich, D; Schnapp, A; Park, JE Design, synthesis, and evaluation of indolinones as inhibitors of the transforming growth factorß receptor I (TGFßRI). J Med Chem 53: 7287-95 (2010)
- Gellibert, F; Woolven, J; Fouchet, MH; Mathews, N; Goodland, H; Lovegrove, V; Laroze, A; Nguyen, VL; Sautet, S; Wang, R; Janson, C; Smith, W; Krysa, G; Boullay, V; De Gouville, AC; Huet, S; Hartley, D Identification of 1,5-naphthyridine derivatives as a novel series of potent and selective TGF-beta type I receptor inhibitors. J Med Chem 47: 4494-506 (2004)
- Robinson, N; Gibson, TM; Chicarelli-Robinson, MI; Cameron, L; Hylands, PJ; Wilkinson, D; Simpson, TJ Cochliobolic acid, a novel metabolite produced by Cochliobolus lunatus, inhibits binding of TGF-alpha to the EGF receptor in a SPA assay. J Nat Prod 60: 6-8 (1997)
- Ciayadi, R; Potdar, M; Walton, KL; Harrison, CA; Kelso, GF; Harris, SJ; Hearn, MT 2-Phenyl and 2-heterocyclic-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridines as inhibitors of TGF-ß1 and activin A signalling. Bioorg Med Chem Lett 21: 5642-5 (2011)
- Yu, WC; Yeh, TY; Ye, CH; Chong, PCT; Ho, YH; So, DK; Yap, KY; Peng, GR; Shao, CH; Jagtap, AD; Chern, JW; Lin, CS; Lin, SP; Lin, SL; Yu, SH; Yu, CW Discovery of HDAC6, HDAC8, and 6/8 Inhibitors and Development of Cell-Based Drug Screening Models for the Treatment of TGF-β-Induced Idiopathic Pulmonary Fibrosis. J Med Chem 66: 10528-10557 (2023)
- Tan, B; Zhang, X; Quan, X; Zheng, G; Li, X; Zhao, L; Li, W; Li, B Design, synthesis and biological activity evaluation of novel 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as potent transforming growth factor-β (TGF-β) type I receptor inhibitors. Bioorg Med Chem Lett 30: (2020)
- Jin, CH; Krishnaiah, M; Sreenu, D; Subrahmanyam, VB; Rao, KS; Lee, HJ; Park, SJ; Park, HJ; Lee, K; Sheen, YY; Kim, DK Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-ß type I receptor kinase as cancer immunotherapeutic/antifibrotic agent. J Med Chem 57: 4213-38 (2014)
- ChEMBL_2303312 Inhibition of TGF-beta (unknown origin)
- ChEMBL_2281209 Inhibition of TGF-beta receptor (unknown origin)
- ChEBML_206339 Inhibitory activity against TGF-beta type I receptor
- ChEMBL_208099 (CHEMBL814455) Dissociation constant for TGF-beta receptor type I
- ChEMBL_305062 (CHEMBL832694) Inhibition of human TGF-beta type II receptor
- ChEMBL_27998 (CHEMBL642728) Inhibition of Activin like receptor kinase 5, TGF beta type I receptor
- ChEMBL_570592 (CHEMBL1030216) Inhibition of TGF-beta-induced ALK5 in human HepG2 cells by luciferase assay
- ChEMBL_946470 (CHEMBL2340067) Inhibition of Alk5 in TGF-beta-stimulated human HepG2 cells assessed as decrease in Smad2 phosphorylation treated for 45 mins prior to TGF-beta stimulation measured after 60 mins by odyssey blot scanner analysis
- ChEMBL_208100 (CHEMBL814456) Inhibition of human Transforming growth factor (TGF) beta-1 receptor (T204D mutation) autophosphorylation in Sf9 cells
- ChEMBL_208101 (CHEMBL814457) In vitro inhibitory activity against human Transforming growth factor beta-1 receptor kinase (TGF-beta RIK)
- ChEMBL_2261292 (CHEMBL5216303) Inhibition of human TGF-beta-R1 using TMB substrate incubated for 2.5 hrs by microplate reader analysis
- ChEMBL_2294673 Inhibition of ALK5 in human Hs-578T cells assessed as effect of TGF-beta-induced Smad3/4 phosphorylation
- ChEMBL_2473490 Inhibition of ALK1/2 in HEK293 cells transfected with CAGA response element luciferase based reporter assessed as inhibition of TGF beta1-induced SMAD-dependent transcriptional activity incubated for 24 hrs in presence of TGF beta1 by dual-luciferase based reporter gene assay
- SMAD Transcription Factor Inhibitors Dose Response Confirmation NIH Molecular Libraries Screening Centers Network [MLSCN] Emory Chemical Biology Discovery Center in MLSCN Assay provider: F.M. Hoffmann, University of Wisconsin-Madison MLSCN Grant: 1R21NS057002-01 Assay Overview: Transforming growth factor beta (TGF-Beta) regulates a variety of processes in mammalian cells, including proliferation, apoptosis, cell migration and extracellular matrix production. Aberrant increases in TGF-Beta signaling have been implicated in several pathological conditions including cancer and fibrosis. Inhibition of TGF-Beta signaling is an important tool in elucidating the multiple biological functions of TGF-Beta and is of significant interest as a potential therapeutic strategy in fibrotic diseases and several advanced cancers. Smad proteins mediate cellular responses to TGF-Beta. TGF-Beta alters cellular gene expression and cell behavior by binding and activating the Type II and Type I serine kinase receptors on the cell membrane. Activated Type I recep
- SMAD Transcription Factor Inhibitors Secondary Dose Response Confirmation NIH Molecular Libraries Screening Centers Network [MLSCN] Emory Chemical Biology Discovery Center in MLSCN Assay provider: F.M. Hoffmann, University of Wisconsin-Madison MLSCN Grant: 1R21NS057002-01 Assay Overview: Transforming growth factor beta (TGF-Beta) regulates a variety of processes in mammalian cells, including proliferation, apoptosis, cell migration and extracellular matrix production. Aberrant increases in TGF-Beta signaling have been implicated in several pathological conditions including cancer and fibrosis. Inhibition of TGF-Beta signaling is an important tool in elucidating the multiple biological functions of TGF-Beta and is of significant interest as a potential therapeutic strategy in fibrotic diseases and several advanced cancers. Smad proteins mediate cellular responses to TGF-Beta. TGF-Beta alters cellular gene expression and cell behavior by binding and activating the Type II and Type I serine kinase receptors on the cell membrane. Activated Type I recep
- ChEMBL_1924659 (CHEMBL4427615) Inhibition of ALK5 in TGF beta1-stimulated HEK293T cells by SMAD binding element-driven beta lactamase reporter gene assay
- ChEMBL_2328042 Inhibition of TGF-beta induced Smad2/3 signaling in human HepG2 cells harboring pRL-EF1alpha, (CAGA)9x-MLP-Luc plasmid assessed as luciferase activity incubated for 24 hrs
- ChEMBL_2436393 Inhibition of ALK5 signaling in mouse NIH3T3 cells transfected with (CAGA)12-luciferase reporter incubated for 18 hrs in presence of TGF-beta1 by dual-luciferase reporter assay
- ChEMBL_2088444 (CHEMBL4769707) Inhibition of TGF-beta type 1 receptor in mouse NIH3T3 cells expressing Luc-Smad 2/3 measured after 24 hrs incubation by cell based Bright-Glo Luciferase assay
- ChEMBL_2161333 (CHEMBL5046083) Inhibition of TGF beta 1 (unknown origin) expressed in HEK293 cells transfected with Smad2/3 responsive reporter plasmid incubated for 4 hrs by dual luciferase reporter gene assay
- ChEMBL_768740 (CHEMBL1831613) Inhibition of TGF-beta1 signaling in human HEK293 cells transfected with luciferase and FAST-2 gene expression vector A3-LUX after 16 hrs by luciferase reporter gene assay
- ChEMBL_2297944 Inhibition of TGF-beta induced Smad2/3 signaling in human HepG2 cells harboring pRL-EF1alpha, (CAGA)9x-MLP-Luc plasmid assessed as luciferase activity incubated for 24 hrs by Promega reporter rene assay
- In Vitro Enzymatic Activity Inhibition Test (TGFbetaR1) (Promega), the inhibitory effect of the compounds of the present invention on the enzymatic activity of TGFβR1 was determined, and the steps were as follows: TGFβR1 enzyme were pre-incubated with different concentrations of test compounds (1000 nM, 100 nM, 10 nM) at 30 C. for 30 min, TGFβR1 peptide and adenosine triphosphate (ATP) were added to initiate the reaction. The incubation was performed at 30 C. for 3 h, followed by an addition of ADP-Glo reagent and incubated at room temperature for 90 min, kinase detection reagent was then added. Chemiluminescence signal values were detected after incubation at room temperature for another 30 min.
- ChEMBL_1994470 (CHEMBL4628365) Inhibition of latency-associated peptide/TGF-beta binding to recombinant human integrin alphaV (Phe31 to Val992 residues) beta6 (Gly22 to Asn707 residues) expressed in CHO cells by ELISA based solid phase binding assay
- ChEMBL_1994471 (CHEMBL4628366) Inhibition of latency-associated peptide/TGF-beta binding to recombinant human integrin alphaV (Phe31 to Val992 residues) beta8 (Glu43 to Arg684 residues) expressed in CHO cells by ELISA based solid phase binding assay
- Biological Assay Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
- Biological Assay Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
- Biological Assay Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
- Biological Assays Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
- Biological Assays Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
- Biological Assay Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
- In-Vitro (Enzyme) Assay In-Vitro (Enzyme) Assay for Determination of the Efficacy of the Inhibitors of the Inhibition of TGF-Beta-Mediated EffectsAs an example, the ability of the inhibitors to eliminate TGF-beta-mediated growth inhibition is tested.Cells of the lung epithelial cell line Mv1Lu are sown in a defined cell density in a 96-well microtitre plate and cultivated overnight under standard conditions. Next day, the medium is replaced by medium which comprises 0.5% of FCS and 1 ng/ml of TGF-beta, and the test substances are added in defined concentrations, generally in the form of dilution series with 5-fold steps. The concentration of the solvent DMSO is constant at 0.5%. After a further two days, Crystal Violet staining of the cells is carried out. After extraction of the Crystal Violet from the fixed cells, the absorption is measured spectrophotometrically at 550 nm. It can be used as a quantitative measure of the adherent cells present and thus of the cell proliferation during the culture.
- In Vitro Enzymatic Activity Inhibition Test (TGFbetaR2) Experimental method: According to the instructions of ADP-Glo Kinase Detection Kit (Promega), the inhibitory effect of the compounds of the present invention on the enzymatic activity of TGFβR2 was determined, and the steps were as follows: TGFβR2 enzyme were pre-incubated with different concentrations of test compounds (1000 nM, 100 nM, 10 nM) at 30 C. for 30 min, myelin basic protein (MBP) and adenosine triphosphate (ATP) were added to initiate the reaction. The incubation was performed at 30 C. for 3 h, followed by an addition of ADP-Glo reagent and incubated at room temperature for 90 min, kinase detection reagent was then added. The chemiluminescence signal values were detected after incubation at room temperature for another 30 min.
- TGFβR1 Kinase Inhibition Assay TGFβR1 kinase assay was performed according to the instruction manual of the ADP-Glo™ Kinase Assay kit provided by Promega. Prepare 1× Kinase buffer (50 mM Tris pH7.5, 0.1% BSA, 10 mM MgCl2, 1 mM DTT). Before activation reaction was started, Compounds were dissolved in DMSO and make 100× solution with 3-fold serial dilution for a total of 10 concentrations. Transfer 50 nL compounds to 384-well plate according to plate map using the automated liquid handler. Prepare enzyme mix containing 2× enzyme mix containing 40 nM TGFβR1 with 1× Kinase buffer, add 2.5 μL enzyme mix to 384-well plate and pre-incubate with compounds at RT for 10 minutes. Prepare 2× substrate mix containing 5.4 μM ATP 1× Kinase buffer and add 2.5 μL substrate mix to 384-well plate, react at RT for 120 min. Add 5 μL ADP-Glo Reagent to terminate the kinase reaction and deplete the remaining ATP, incubate at RT for 60 minutes. Add 10 μL Kinase Detection Reagent to convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferin reaction, incubate at RT for 30 minutes. Collect luminescence data with Envision.
- Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
- In vitro Assay The enzymatic activity of compounds of the present invention was monitored measuring the formation of ADP using the ADP-GLO Kinases assay. Following the incubation of the purified enzyme, a substrate and ATP, the produced ADP was converted into ATP, which in turn was converted into light by Ultra-Glo Luciferase. The luminescent signal positively correlated with ADP amount and kinase activity. Briefly, the kinase reaction was performed by incubating 2.6 nM of the purified, commercially available human ALK5 (recombinant TGF (31 N-term GST-tagged, 80-end), a final concentration of TGF(31 peptide 94.5 μM (Promega, T36-58) and ultra-pure ATP (Promega V915B). The ATP concentration was set at the Km value (concentration of substrate which permits the enzyme to achieve half maximal velocity (Vmax)) of ALK5 (5 μM). All reactions/incubations were performed at 25° C. Compound and ALK5 kinase were mixed and incubated for 15 mins. Reactions were initiated by addition of ATP at a final concentration in the assay of 0.8301 After an incubation of 150 min, the reaction was stopped, and ADP production detected with ADP-Glo kit according to manufacturer's indications.
- in vitro Kinase Assay Shown are the IC50s (concentrations causing 50% inhibition) of DM and the analogues for the in vitro kinase assays using the following purified human enzymes: the BMP type-I receptor activin receptor-like kinase 2 (ALK2/BMPR-I), the TGFβ type-I receptor activin receptor-like kinase 5 (ALK5/ TGFβR-I), the VEGF type-2 receptor (VEGFR2/KDR), the AMP-activated protein kinase (AMPK), and the platelet-derived growth factor receptor-β (PDGFR β). In in vitro kinase assays, DM was relatively nonspecific, targeting ALK2, AMPK, and KDR with IC50s of <250 nM. LDN-193189 was slightly more selective but still had significant effects against ALK5 and KDR. By comparison, DMH1, DMH2, and DMH3 were much more selective ALK2 inhibitors. In particular, DMH1 had no detectible activity against any of the kinases tested besides ALK2. DMH4 was a selective KDR inhibitor with modest effect on ALK2 (IC50 3.6 uM) and minimal effect on AMPK (IC50 8.0 uM). Nonspecific kinase inhibitor staurosporine was used as a control. All of the reactions were carried out in the presence of 10 uM ATP.
- TGF-beta 1 Assay Compound Screening: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:2, 11-point dose-response curves from 10 μM to 1.87 nM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well clear bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. LL29 cells were plated at 1,500 cells/well in 80 μl/well F12 medium supplemented with 1% Fetal Bovine Serum. One hour after addition of the cells, TGF-β1 (Peprotech; 20 ng/mL) was added to the plates to induce fibrosis (ref. 1 and 2 above). Wells treated with TGF-β1 and containing DMSO were used as controls. Cells were incubated at 37° C. and 5% CO2 for 4 days. Following incubation for 4 days, SYTOX green nucleic acid stain (Life Technologies [Thermo Fisher Scientific]) was added to the wells at a final concentration of 1 uM and incubated at room temperature for 30 min. Cells were then fixed using 4% formaldehyde (Electron Microscopy Sciences), washed 3 times with PBS followed by blocking and permeabilization using 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS. Cells were then stained with antibody specific to α-smooth muscle actin (aSMA; Abcam) (ref. 1 and 2 above) in 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS, and incubated overnight at 4° C. Cells were then washed 3 times with PBS, followed by incubation with Alexa Flor-647 conjugated secondary antibody (Life Technologies [Thermo Fisher Scientific]) and DAPI at room temperature for 1 hour. Cells were then washed 3 times with PBS and plates were sealed for imaging. αSMA staining was imaged by excitation at 630 nm and emission at 665 nm and quantified using the Compartmental Analysis program on the CellInsight CX5 (Thermo Scientific). Dead or apoptotic cells were excluded from analysis based on positive SYTOX green staining. % of total cells positive for αSMA were counted in each well and normalized to the average of 11 wells treated with TGF-β1 on the same plate using Dotmatics' Studies Software. The normalized averages (fold change over untreated) of 3 replicate wells for each compound concentration were used to create dose-responses curves and EC50 values were calculated using non-linear regression curve fit in the Dotmatics' Studies Software.
- Enzymatic Activity Assay The enzymatic activity of compounds of the present invention was monitored measuring the formation of ADP using the ADP-GLO Kinases assay. Following the incubation of the purified enzyme, a substrate and ATP, the produced ADP was converted into ATP, which in turn was converted into light by Ultra-Glo Luciferase. The luminescent signal positively correlated with ADP amount and kinase activity. Briefly, the kinase reaction was performed by incubating 2.6 nM of the purified, commercially available human ALK5 (recombinant TGF β1 N-term GST-tagged, 80-end), a final concentration of TGFβ1 peptide 94.5 μM (Promega, T36-58) and ultra-pure ATP (Promega V915B). The ATP concentration was set at the Km value (concentration of substrate which permits the enzyme to achieve half maximal velocity (Vmax)) of ALK5 (5 μM). All reactions/incubations were performed at 25° C. Compound and ALK5 kinase were mixed and incubated for 15 mins. Reactions were initiated by addition of ATP at a final concentration in the assay of 0.83 μM. After an incubation of 150 min, the reaction was stopped, and ADP production detected with ADP-Glo kit according to manufacturer's indications. The assay was performed in 384-well format and was validated using a selection of reference compounds that was tested in 11 point concentration-response curve.
- In Vitro Assay The enzymatic activity of compounds of the present invention was monitored measuring the formation of ADP using the ADP-GLO Kinases assay. Following the incubation of the purified enzyme, a substrate and ATP, the produced ADP was converted into ATP, which in turn was converted into light by Ultra-Glo Luciferase. The luminescent signal positively correlated with ADP amount and kinase activity. Briefly, the kinase reaction was performed by incubating 2.6 nM of the purified, commercially available human ALK5 (recombinant TGF 31 N-term GST-tagged, 80-end), a final concentration of TGFβ1 peptide 94.5 μM (Promega, T36-58) and ultra-pure ATP (Promega V915B). The ATP concentration was set at the Km value (concentration of substrate which permits the enzyme to achieve half maximal velocity (Vmax)) of ALK5 (5 μM). All reactions/incubations were performed at 25° C. Compound and ALK5 kinase were mixed and incubated for 15 mins. Reactions were initiated by addition of ATP at a final concentration in the assay of 0.83p M. After an incubation of 150 min, the reaction was stopped, and ADP production detected with ADP-Glo kit according to manufacturer's indications. The assay was performed in 384-well format and was validated using a selection of reference compounds that was tested in 11 point concentration-response curve.
- Inhibition Assay TGFβRI kinase assay kit (V4093, Promega) was used to assay enzyme activity. 2 μl of enzyme solution (the final concentration of enzyme in the reaction system was 2 ng/μL) formulated with reaction buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA), 1 μl of a 3-fold gradient dilution of the compounds dissolved in 5% DMSO, and 2 μl of a mixed solution of ATP and TGFβRI substrate peptide (the final concentration of ATP was 50 μM, and the final concentration of substrate was 0.2 μg/μL) were added successively to a 384-well plate (4514, Corning). After reaction at 27° C. for 2.5 hours, 5 μl of ADP-Glo solution in the kit was added to each well, then the plate was placed at 27° C. for 40 minutes. 10 μl of kinase assay reagent was then added to each well, then the plate was placed at 27° C. for 30 minutes. The chemiluminescence signal values were measured with a Victor 3 (PerkinElmer) multi-function microplate reader. The IC50 values of the compounds for enzyme inhibition were calculated using Graphpad prism software based on each concentration of the compound and the corresponding signal value thereof.
- Kinase Activity Assay TGFβRI kinase assay kit (V4093, Promega) was used to assay enzyme activity. 2 μl of enzyme solution (the final concentration of enzyme in the reaction system was 2 ng/μL) formulated with reaction buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA), 1 μl of a 3-fold gradient dilution of the compounds dissolved in 5% DMSO, and 2 μl of a mixed solution of ATP and TGFβRI substrate peptide (the final concentration of ATP was 50 μM, and the final concentration of substrate was 0.2 μg/μL) were added successively to a 384-well plate (4514, Corning). After reaction at 27° C. for 2.5 hours, 5 μl of ADP-Glo solution in the kit was added to each well, then the plate was placed at 27° C. for 40 minutes. 10 μl of kinase assay reagent was then added to each well, then the plate was placed at 27° C. for 30 minutes. The chemiluminescence signal values were measured with a Victor 3 (PerkinElmer) multi-function microplate reader. The IC50 values of the compounds for enzyme inhibition were calculated using Graphpad prism software based on each concentration of the compound and the corresponding signal value thereof.
- Inhibition of fibrosis Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 8-point dose-response curves from 15 μM to 5 nM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well clear bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. LL29 cells were plated at 1,000 cells/well in 50 μl/well serum free F12 medium. One hour after addition of the cells, TGF-β1 (Peprotech; 10 ng/ml) was added to the plates to induce fibrosis (ref 1 and 2 above). Wells untreated with TGF-β1 were used as control for normalization and calculating IC50 values. Cells were incubated at 37° C. and 5% CO2 for 3 days. Cells were fixed using 4% formaldehyde (Electron Microscopy Sciences), washed 3 times with PBS followed by blocking and permeabilization using 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS. Cells were then stained with antibody specific to α-smooth muscle actin (αSMA; Abcam) (ref. 1 and 2 above) in 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS, and incubated overnight at 4° C. Cells were then washed 3 times with PBS, followed by incubation with Alexa Flor-647 conjugated secondary antibody (Life Tech) and DAPI at room temperature for 1 hour. Cells were then washed 3 times with PBS and plates were sealed for imaging. αSMA staining was imaged by excitation at 630 nm and emission at 665 nm and quantified using the Compartmental Analysis program on the CellInsight CX5 (Thermo Scientific). % of total cells positive for αSMA were counted in each well and normalized to the average of 8 wells treated with TGF-β1 on the same plate using Excel (Microsoft Inc.). The normalized averages (fold change over untreated) of 6 replicate wells for each compound concentration were used to create dose-responses curves and IC50 values were calculated using non-linear regression curve fit in Prism (GraphPad).
- Binding Assay Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Binding Assay Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Solid Phase Integrin alphavbeta6 Binding Assay Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C. overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Solid Phase Integrin αVβ6 Binding Assay Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25 OC, overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Solid Phase Integrin alphaVbeta6 Binding Assay Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Solid Phase Integrin alphav/beta6 Binding Assay (B-1) Microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by four-parameter logistic regression.
- Solid Phase Integrin alphavbeta6 Binding Assay Microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- In Vitro Kinase Assay - Inhibition of ALK1/2/3/4/5/6 Kinases Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle.
- In Vitro Kinase Assay - Inhibition of ALK1/2/3/4/5/6 Kinases Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ3 family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle.
- In Vitro Kinase Assay Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle.
- Signal Transduction Inhibitory Assay The biological activity of a compound was determined by measuring the activation of Smad3/Smad4 complex which is a transcription factor showing activation induced by TGF-beta stimulation. That is, a reporter plasmid having a DNA sequence (CAGA sequence), which is activated by binding to Smad3/Smad4 complex, is linked to the upstream of the luciferase gene (luminescence enzyme) was prepared. Mink lung epithelial cells CCL64 (named as x9CAGA/CCL64 cells) stably incorporating this reporter plasmid were established. The x9CAGA/CCL64 cells were cultured in DMEM medium containing 10% FBS, penicillin (100 U/ml), streptomycin (100 g/ml), and blasticidin S (1 ug/ml). The x9CAGA/CCL64 cells were seeded in a 96 well plate at a concentration of 10000 cells/well, and cultured in a 5% CO2 incubator at 37C. The next day, the medium was changed to DMEM medium containing 0.2% FBS, and the test compound was added.
- In Vitro Kinase Assay Inhibition of ALK1/2/3/4/5/6 Kinases Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle. IC50 values were generated from activity values performed at multiple concentrations.
- Solid Phase Integrin alphav/beta6 Binding Assay (B-2) A third series of exemplary compounds was selected for testing in the solid phase integrin αvβ6 binding assay. The compounds tested were compound samples prepared according to procedures described in the Synthetic Examples section, with the stereochemical purity as indicated in the Examples. As in Example B1, microplates were coated with recombinant human integrin αvβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
- Human TH17 Cytokine Inhibition ELISA Assay Peripheral blood mononuclear cells (PBMCs) were sourced from freshly prepared leukocyte enriched plasma (buffy coat) from healthy donors (New York Blood Center). PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Human CD4+ T cells were seeded into 96-well plates (5×104 cells/well) and activated with plate-bound anti-human (h)-CD3 antibody and soluble h-aCD28 (both at 1 ug/ml; eBioscience) and differentiated into TH17 cells with 20 ng/mL h-IL-6, 5 ng/mL h-TGF-β1, 10 ng/mL h-IL-23 (eBioscience) and 10 ng/mL IL-1 β (Miltenyi Biotec) in serum-free TexMACS Medium (Miltenyi Biotec) supplemented with 1% Penicillin/Streptomycin (Lonza) for 3 days. CD4+ T cells propagated under TH17-polarizing conditions were cultured in the presence or absence of various concentrations of compounds with a final concentration of 0.1% DMSO. Supernatants were collected and stored at −20° C. until assayed for IL-17A, IL-17F and IL-21 levels by Ready-Set-Go ELISA kits (eBioscience) as per manufacturer's instructions. Endpoint absorbance was read at 450 nm using a microplate reader (Perkin Elmer). The half maximal inhibitory concentrations (IC50) for representative compounds of the invention were determined by GraphPad Prism software.
- Binding Assay The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN , Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
- Human Th17 Assay The human Th17 assay tests the effect of RORγt modulatory compounds on IL-17 production by CD4 T cells under conditions which favor Th17 differentiation. Total CD4+ T cells were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors using a CD4+ T cell isolation kit II, following the manufacturer's instructions (Miltenyi Biotec). Cells were resuspended in a medium of RPMI-1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, glutamate, and β-mercaptoethanol and were added to 96-well plates at 1.5×105 per 100 μL per well. 50 μL of compound at titrated concentrations in DMSO were added into each well at final DMSO concentration at 0.2%. Cells were incubated for 1 hour, then 50 μL of Th17 cell differentiation medium was added to each well. The final concentrations of antibodies and cytokines (R&D Systems) in differentiation medium were: 3×106/mL anti-CD3/CD28 beads (prepared using human T cell activation/expansion kit, Miltenyi Biotec), 10 μg/mL anti-IL4, 10 μg/mL anti-IFNγ, 10 ng/mL IL13, 10 ng/mL IL23, 50 ng/mL IL6, 3 ng/mL TGFβ and 20 U/mL IL2. Cells were cultured at 37° C. and 5% CO2 for 3 days. Supernatants were collected and the accumulated IL-17 in culture was measured by using MULTI-SPOT Cytokine Plate following manufacture's instruction (Meso Scale Discovery). The plate was read using Sector Imager 6000, and IL-17 concentration was extrapolated from the standard curve.