- Seifert, K; Büttner, A; Rigol, S; Eilert, N; Wandel, E; Giannis, A Potent small molecule Hedgehog agonists induce VEGF expression in vitro. Bioorg Med Chem 20: 6465-81 (2012)
- Musumeci, F; Radi, M; Brullo, C; Schenone, S Vascular endothelial growth factor (VEGF) receptors: drugs and new inhibitors. J Med Chem 55: 10797-822 (2012)
- Kiselyov, AS; Semenova, M; Semenov, VV 3,4-Disubstituted isothiazoles: novel potent inhibitors of VEGF receptors 1 and 2. Bioorg Med Chem Lett 19: 1195-8 (2009)
- Manley, PW; Furet, P; Bold, G; Brüggen, J; Mestan, J; Meyer, T; Schnell, CR; Wood, J; Haberey, M; Huth, A; Krüger, M; Menrad, A; Ottow, E; Seidelmann, D; Siemeister, G; Thierauch, KH Anthranilic acid amides: a novel class of antiangiogenic VEGF receptor kinase inhibitors. J Med Chem 45: 5687-93 (2002)
- Kiselyov, AS; Semenova, M; Semenov, VV; Piatnitski, E; Ouyang, S Hetaryl imidazoles: a novel dual inhibitors of VEGF receptors I and II. Bioorg Med Chem Lett 16: 1440-4 (2006)
- Curtin, ML; Frey, RR; Heyman, HR; Soni, NB; Marcotte, PA; Pease, LJ; Glaser, KB; Magoc, TJ; Tapang, P; Albert, DH; Osterling, DJ; Olson, AM; Bouska, JJ; Guan, Z; Preusser, LC; Polakowski, JS; Stewart, KD; Tse, C; Davidsen, SK; Michaelides, MR Thienopyridine ureas as dual inhibitors of the VEGF and Aurora kinase families. Bioorg Med Chem Lett 22: 3208-12 (2012)
- García-Aranda, MI; Marrero, P; Gautier, B; Martín-Martínez, M; Inguimbert, N; Vidal, M; García-López, MT; Jiménez, MA; González-Muñiz, R; Pérez de Vega, MJ Parallel solid-phase synthesis of a small library of linear and hydrocarbon-bridged analogues of VEGF(81-91): potential biological tools for studying the VEGF/VEGFR-1 interaction. Bioorg Med Chem 19: 1978-86 (2011)
- Bilodeau, MT; Cunningham, AM; Koester, TJ; Ciecko, PA; Coll, KE; Huckle, WR; Hungate, RW; Kendall, RL; McFall, RC; Mao, X; Rutledge, RZ; Thomas, KA Design and synthesis of 1,5-diarylbenzimidazoles as inhibitors of the VEGF-receptor KDR. Bioorg Med Chem Lett 13: 2485-8 (2003)
- Wu, Z; Fraley, ME; Bilodeau, MT; Kaufman, ML; Tasber, ES; Balitza, AE; Hartman, GD; Coll, KE; Rickert, K; Shipman, J; Shi, B; Sepp-Lorenzino, L; Thomas, KA Design and synthesis of 3,7-diarylimidazopyridines as inhibitors of the VEGF-receptor KDR. Bioorg Med Chem Lett 14: 909-12 (2004)
- Han, SY; Park, SS; Lee, WG; Min, YK; Kim, BT Synthesis of a novel biotin-tagged photoaffinity probe for VEGF receptor tyrosine kinases. Bioorg Med Chem Lett 16: 129-33 (2005)
- Puszko, AK; Sosnowski, P; Pułka-Ziach, K; Hermine, O; Hopfgartner, G; Lepelletier, Y; Misicka, A Urea moiety as amide bond mimetic in peptide-like inhibitors of VEGF-A Bioorg Med Chem Lett 29: 2493-2497 (2019)
- Kiselyov, AS; Semenova, M; Semenov, VV (1,2,3-Triazol-4-yl)benzenamines: synthesis and activity against VEGF receptors 1 and 2. Bioorg Med Chem Lett 19: 1344-8 (2009)
- Hughes, TV; Emanuel, SL; Beck, AK; Wetter, SK; Connolly, PJ; Karnachi, P; Reuman, M; Seraj, J; Fuentes-Pesquera, AR; Gruninger, RH; Middleton, SA; Lin, R; Davis, JM; Moffat, DF 4-Aryl-5-cyano-2-aminopyrimidines as VEGF-R2 inhibitors: synthesis and biological evaluation. Bioorg Med Chem Lett 17: 3266-70 (2007)
- Hou, F; Yao, Y; Wei, Y; Wang, Y; Cao, Y; Liu, X; Zheng, L; Zhang, Q; Jiao, Y; Chen, Y; Meng, Y; Sun, Y; Wu, Y; Wang, J; Wang, J; Wu, Z; Zhang, K; Wei, M; Yang, G Design and discovery of new selective and potent VEGF receptor 2 tyrosine kinase inhibitors. Bioorg Med Chem 91: (2023)
- Kiselyov, AS; Milligan, D; Ouyang, X Novel inhibitors of VEGF receptors-1 and -2 based on azole-5-carboxamide templates. Bioorg Med Chem Lett 17: 3550-7 (2007)
- Kiselyov, AS; Piatnitski, EL; Samet, AV; Kisliy, VP; Semenov, VV ortho-Substituted azoles as selective and dual inhibitors of VEGF receptors 1 and 2. Bioorg Med Chem Lett 17: 1369-75 (2007)
- Hegde, VR; Pu, H; Patel, M; Jachens, A; Gullo, VP; Chan, TM A new antitumor compound from the plant Oryctanthus sp. as a VEGF receptor binding inhibitor. Bioorg Med Chem Lett 15: 4907-9 (2005)
- Hao, J; Ho, JN; Lewis, JA; Karim, KA; Daniels, RN; Gentry, PR; Hopkins, CR; Lindsley, CW; Hong, CC In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors. ACS Chem Biol 5: 245-53 (2010)
- Jarvis, A; Allerston, CK; Jia, H; Herzog, B; Garza-Garcia, A; Winfield, N; Ellard, K; Aqil, R; Lynch, R; Chapman, C; Hartzoulakis, B; Nally, J; Stewart, M; Cheng, L; Menon, M; Tickner, M; Djordjevic, S; Driscoll, PC; Zachary, I; Selwood, DL Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction. J Med Chem 53: 2215-26 (2010)
- Baindur, N; Chadha, N; Brandt, BM; Asgari, D; Patch, RJ; Schalk-Hihi, C; Carver, TE; Petrounia, IP; Baumann, CA; Ott, H; Manthey, C; Springer, BA; Player, MR 2-Hydroxy-4,6-diamino-[1,3,5]triazines: a novel class of VEGF-R2 (KDR) tyrosine kinase inhibitors. J Med Chem 48: 1717-20 (2005)
- Hennequin, LF; Thomas, AP; Johnstone, C; Stokes, ES; Plé, PA; Lohmann, JJ; Ogilvie, DJ; Dukes, M; Wedge, SR; Curwen, JO; Kendrew, J; Lambert-van der Brempt, C Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J Med Chem 42: 5369-89 (1999)
- Kiselyov, AS; Piatnitski, E; Semenova, M; Semenov, VV N-(Aryl)-4-(azolylethyl)thiazole-5-carboxamides: novel potent inhibitors of VEGF receptors I and II. Bioorg Med Chem Lett 16: 602-6 (2006)
- Lücking, U; Lücking, M; Krüger, R; Siemeister, G Sulfoximine-substituted pyrimidines as CDK- and/or VEGF inhibitors, their production and use as pharmaceutical agents US Patent US8507510 (2013)
- Nosaka, C; Adachi, H; Sawa, R; Nakae, K; Atsumi, S; Kinoshita, N; Kubota, Y; Igarashi, M; Sei, Y; Yamaguchi, K; Shibuya, M; Nishimura, Y; Akamatsu, Y Vegfrecine, an inhibitor of VEGF receptor tyrosine kinases isolated from the culture broth of Streptomyces sp. J Nat Prod 76: 715-9 (2013)
- Hudkins, RL; Zulli, AL; Underiner, TL; Angeles, TS; Aimone, LD; Meyer, SL; Pauletti, D; Chang, H; Fedorov, EV; Almo, SC; Fedorov, AA; Ruggeri, BA 8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors. Bioorg Med Chem Lett 20: 3356-60 (2010)
- Peifer, C; Bühler, S; Hauser, D; Kinkel, K; Totzke, F; Schächtele, C; Laufer, S Design, synthesis and characterization of N9/N7-substituted 6-aminopurines as VEGF-R and EGF-R inhibitors. Eur J Med Chem 44: 1788-93 (2009)
- Kiselyov, AS; Semenova, M; Semenov, VV; Milligan, D Inhibitors of VEGF receptors-1 and -2 based on the 2-((pyridin-4-yl)ethyl)pyridine template. Bioorg Med Chem Lett 16: 1913-9 (2006)
- Falcon, BL; Barr, S; Gokhale, PC; Chou, J; Fogarty, J; Depeille, P; Miglarese, M; Epstein, DM; McDonald, DM Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors. Cancer Res 71: 1573-83
- Coothankandaswamy, V; Liu, Y; Mao, SC; Morgan, JB; Mahdi, F; Jekabsons, MB; Nagle, DG; Zhou, YD The alternative medicine pawpaw and its acetogenin constituents suppress tumor angiogenesis via the HIF-1/VEGF pathway. J Nat Prod 73: 956-61 (2010)
- Goff, D; Zhang, J; Heckrodt, T; Yu, J; Ding, P; Singh, R; Holland, S; Li, W; Irving, M Discovery of dual Axl/VEGF-R2 inhibitors as potential anti-angiogenic and anti-metastatic drugs for cancer chemotherapy. Bioorg Med Chem Lett 27: 3766-3771 (2017)
- Kwon, HS; Kim, DR; Yang, EG; Park, YK; Ahn, HC; Min, SJ; Ahn, DR Inhibition of VEGF transcription through blockade of the hypoxia inducible factor-1α-p300 interaction by a small molecule. Bioorg Med Chem Lett 22: 5249-52 (2012)
- Duncton, MA; Piatnitski, EL; Katoch-Rouse, R; Smith, LM; Kiselyov, AS; Milligan, DL; Balagtas, C; Wong, WC; Kawakami, J; Doody, JF Arylphthalazines. Part 2: 1-(Isoquinolin-5-yl)-4-arylamino phthalazines as potent inhibitors of VEGF receptors I and II. Bioorg Med Chem Lett 16: 1579-81 (2006)
- Udugamasooriya, DG; Ritchie, C; Brekken, RA; Kodadek, T A peptoid antagonist of VEGF receptor 2 recognizes a 'hotspot' in the extracellular domain distinct from the hormone-binding site. Bioorg Med Chem 16: 6338-43 (2008)
- Underiner, TL; Ruggeri, B; Aimone, L; Albom, M; Angeles, T; Chang, H; Hudkins, RL; Hunter, K; Josef, K; Robinson, C; Weinberg, L; Yang, S; Zulli, A TIE-2/VEGF-R2 SAR and in vitro activity of C3-acyl dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole analogs. Bioorg Med Chem Lett 18: 2368-72 (2008)
- Bold, G; Altmann, KH; Frei, J; Lang, M; Manley, PW; Traxler, P; Wietfeld, B; Bruggen, J; Buchdunger, E; Cozens, R; Ferrari, S; Furet, P; Hofmann, F; Martiny-Baron, G; Mestan, J; Rosel, J; Sills, M; Stover, D; Acemoglu, F; Boss, E; Emmenegger, R; Lasser, L; Masso, E; Roth, R; Schlachter, C; Vetterli, W New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis. J Med Chem 43: 2310-23 (2000)
- Hughes, TV; Emanuel, SL; O'Grady, HR; Connolly, PJ; Rugg, C; Fuentes-Pesquera, AR; Karnachi, P; Alexander, R; Middleton, SA 7-[1H-Indol-2-yl]-2,3-dihydro-isoindol-1-ones as dual Aurora-A/VEGF-R2 kinase inhibitors: design, synthesis, and biological activity. Bioorg Med Chem Lett 18: 5130-3 (2008)
- Dandu, R; Zulli, AL; Bacon, ER; Underiner, T; Robinson, C; Chang, H; Miknyoczki, S; Grobelny, J; Ruggeri, BA; Yang, S; Albom, MS; Angeles, TS; Aimone, LD; Hudkins, RL Design and synthesis of dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole oximes as potent dual inhibitors of TIE-2 and VEGF-R2 receptor tyrosine kinases. Bioorg Med Chem Lett 18: 1916-21 (2008)
- Becknell, NC; Zulli, AL; Angeles, TS; Yang, S; Albom, MS; Aimone, LD; Robinson, C; Chang, H; Hudkins, RL Novel C-3 N-urea, amide, and carbamate dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazole analogs as potent TIE-2 and VEGF-R2 dual inhibitors. Bioorg Med Chem Lett 16: 5368-72 (2006)
- Hennequin, LF; Stokes, ES; Thomas, AP; Johnstone, C; Ple, PA; Ogilvie, DJ; Dukes, M; Wedge, SR; Kendrew, J; Curwen, JO Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J Med Chem 45: 1300-12 (2002)
- Peifer, C; Krasowski, A; Hämmerle, N; Kohlbacher, O; Dannhardt, G; Totzke, F; Schächtele, C; Laufer, S Profile and molecular modeling of 3-(indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1 H-pyrrole-2,5-dione (1) as a highly selective VEGF-R2/3 inhibitor. J Med Chem 49: 7549-53 (2006)
- Sun, L; Tran, N; Liang, C; Tang, F; Rice, A; Schreck, R; Waltz, K; Shawver, LK; McMahon, G; Tang, C Design, synthesis, and evaluations of substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF receptor tyrosine kinases. J Med Chem 42: 5120-30 (1999)
- Peifer, C; Selig, R; Kinkel, K; Ott, D; Totzke, F; Schächtele, C; Heidenreich, R; Röcken, M; Schollmeyer, D; Laufer, S Design, synthesis, and biological evaluation of novel 3-aryl-4-(1H-indole-3yl)-1,5-dihydro-2H-pyrrole-2-ones as vascular endothelial growth factor receptor (VEGF-R) inhibitors. J Med Chem 51: 3814-24 (2008)
- Vieth, M; Cummins, DJ DoMCoSAR: a novel approach for establishing the docking mode that is consistent with the structure-activity relationship. Application to HIV-1 protease inhibitors and VEGF receptor tyrosine kinase inhibitors. J Med Chem 43: 3020-32 (2000)
- Egert-Schmidt, AM; Dreher, J; Dunkel, U; Kohfeld, S; Preu, L; Weber, H; Ehlert, JE; Mutschler, B; Totzke, F; Schächtele, C; Kubbutat, MH; Baumann, K; Kunick, C Identification of 2-anilino-9-methoxy-5,7-dihydro-6H-pyrimido[5,4-d][1]benzazepin-6-ones as dual PLK1/VEGF-R2 kinase inhibitor chemotypes by structure-based lead generation. J Med Chem 53: 2433-42 (2010)
- Sun, L; Tran, N; Liang, C; Hubbard, S; Tang, F; Lipson, K; Schreck, R; Zhou, Y; McMahon, G; Tang, C Identification of substituted 3-[(4,5,6, 7-tetrahydro-1H-indol-2-yl)methylene]-1,3-dihydroindol-2-ones as growth factor receptor inhibitors for VEGF-R2 (Flk-1/KDR), FGF-R1, and PDGF-Rbeta tyrosine kinases. J Med Chem 43: 2655-63 (2000)
- Hudkins, RL; Becknell, NC; Zulli, AL; Underiner, TL; Angeles, TS; Aimone, LD; Albom, MS; Chang, H; Miknyoczki, SJ; Hunter, K; Jones-Bolin, S; Zhao, H; Bacon, ER; Mallamo, JP; Ator, MA; Ruggeri, BA Synthesis and biological profile of the pan-vascular endothelial growth factor receptor/tyrosine kinase with immunoglobulin and epidermal growth factor-like homology domains 2 (VEGF-R/TIE-2) inhibitor 11-(2-methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c J Med Chem 55: 903-13 (2012)
- VEGF-PLAP Placental alkaline phosphatase (PLAP) reporter assay controlled by VEGF promoter (VEGF-PLAP)
- ChEMBL_2367963 Inhibition of VEGF (unknown origin)
- ELISA Assay ELISA assay using VEGF.
- ChEBML_214500 Inhibition of VEGF-receptor 2 (KDR)
- ChEMBL_2276620 Inhibition of VEGF-R2 (unknown origin)
- ChEMBL_1449896 (CHEMBL3380585) Inhibition of HIF1 signaling in human U251 cells expressing VEGF by VEGF promoter-driven PLAP reporter gene assay
- ChEMBL_2477378 Inhibition of VEGF (unknown origin) by FRET based analysis
- ChEMBL_635313 (CHEMBL1119626) Inhibition of VEGF-stimulated VEGFR2 autophosphorylation in HUVEC
- ChEMBL_613684 (CHEMBL1070927) Inhibition of VEGFR2 in HUE cells assessed as inhibition of VEGF-induced autophosphorylation treated for 90 mins before VEGF challenge by ELISA
- ChEMBL_840551 (CHEMBL2090016) Inhibition of human KDR phosphorylation expressed in VEGF-stimulated HUVEC treated 90 mins before VEGF stimulation measured after 5 mins by ELISA
- ChEMBL_1615837 (CHEMBL3857906) Inhibition of VEGF induced VEGFR2 phosphorylation in HUVEC preincubated for 1 hr followed by VEGF-stimulation for 5 mins by Western blot analysis
- ChEMBL_1927641 (CHEMBL4430713) Inhibition of biotinylated VEGF-A165 binding to recombinant KDR (unknown origin) measured after 2 hrs by competition binding assay relative to VEGF-A165
- ChEMBL_2504847 Inhibition of KDR phosphorylation in VEGF-induced HEK293 cells preincubated with compound for 60 mins followed by VEGF-induction and measured after 8 mins
- ChEMBL_325520 (CHEMBL859219) Binding affinity for VEGF by BIAcore solution affinity assay
- ChEMBL_1657722 (CHEMBL4007192) Inhibition of VEGF-induced VEGFR2 phosphorylation in human U251 cells preincubated for 60 mins followed by VEGF induction measured after 10 mins by ELISA
- ChEMBL_1778843 (CHEMBL4235835) Inhibition of VEGF-induced VEGFR2 activation in human U251 cells pretreated for 60 mins followed by VEGF addition and measured after 10 mins by ELISA
- ChEMBL_807901 (CHEMBL1959284) Inhibition of VEGF-induced VEGFR2 autophosphorylation in human U251 cells incubated for 60 mins prior to VEGF-induction measured after 10 mins by phosphotyrosine ELISA
- ChEMBL_807902 (CHEMBL1959285) Inhibition of VEGF-induced VEGFR1 autophosphorylation in human A498 cells incubated for 60 mins prior to VEGF-induction measured after 10 mins by phosphotyrosine ELISA
- ChEMBL_312258 (CHEMBL837258) Inhibition of VEGF-stimulated human umbilical vein endothelial cell proliferation
- ChEMBL_476042 (CHEMBL928168) Inhibition of VEGF-induced KDR phosphorylation in mouse 3T3 cells
- ChEMBL_631828 (CHEMBL1111899) Inhibition of VEGF binding to NRP1 expressed in CHO cells
- Inhibition Assay Inhibition of VEGF-stimulated VEGFR-2 autophosphorylation in intact cells.
- ChEMBL_1706544 (CHEMBL4057777) Inhibition of VEGF stimulated VEGFR2 phosphorylation at Y165 residues in HUVEC preincubated for 1 hr followed by VEGF addition measured after 5 mins by Western blot analysis
- ChEMBL_464752 (CHEMBL930118) Inhibition of VEGF-induced human KDR phosphorylation in mouse 3T3 cells
- ChEMBL_565918 (CHEMBL960813) Inhibition of VEGF-induced phosphorylation of VEGFR2 by cell-based ELISA
- ChEMBL_610687 (CHEMBL1072348) Binding affinity to VEGF by surface plasmon-based solution affinity assay
- ChEMBL_617640 (CHEMBL1101249) Displacement of biotinylated-VEGF-A125 from human NRP1 expressed in HUVEC
- ChEMBL_946793 (CHEMBL2344597) Inhibition of VEGFR2 in human U251 cells assessed as inhibition of VEGF-induced tyrosine phosphorylation incubated for 60 mins prior to VEGF-activation measured 10 mins by ELISA
- ChEMBL_841303 (CHEMBL2090044) Inhibition of KDR in human HUVEC cells assessed as inhibition of VEGF-induced ERK phosphorylation incubated for 1 hr prior to VEGF-stimulation measured after 5 mins by ELISA
- ChEMBL_1290299 (CHEMBL3118621) Inhibition of PHD2 in HEK293 cells assessed as VEGF release by immunoassay
- ChEMBL_1448512 (CHEMBL3379264) Inhibition of biotinylated VEGF-A165 from rat recombinant NRP-1 Fc domain
- ChEMBL_2427257 Inhibition of [125I]-VEGF-A165 binding to NRP1 (unknown origin) in HUVEC cells
- ChEMBL_326142 (CHEMBL862697) Inhibitory activity against VEGF stimulated autophosphorylation of VEGFR2 expressed in KDR15 cells
- ChEMBL_840112 (CHEMBL2090815) Binding affinity to VEGF by surface plasmon resonance-based solution affinity assay
- ChEMBL_1649109 (CHEMBL3998243) Inhibition of VEGFR-2 in human U251 cells assessed as reduction in VEGF induced phosphorylation preincubated for 60 mins followed by VEGF addition measured after 10 mins by ELISA method
- ChEMBL_946707 (CHEMBL2343567) Inhibition of VEGF-induced VEGFR2 phosphorylation in human U251 cells overexpressing Flk-1 pretreated for 60 mins prior to VEGF addition measured after 10 mins by phosphotyrosine ELISA cytoblot analysis
- ChEMBL_946708 (CHEMBL2343568) Inhibition of VEGF-induced VEGFR1 phosphorylation in human A498 cells overexpressing Flt-1 pretreated for 60 mins prior to VEGF addition measured after 10 mins by phosphotyrosine ELISA cytoblot analysis
- ChEMBL_2292137 Inhibition of VEGF-induced human VEGFR2 autophosphorylation transfected in CHO cells by chemiluminescence assay
- ChEMBL_380316 (CHEMBL864892) Inhibition of VEGF-induced human KDR phosphorylation transfected in NIH3T3 cells by ELISA
- ChEMBL_429059 (CHEMBL920442) Inhibition of VEGF-induced human KDR phosphorylation in mouse 3T3 cells by ELISA
- ChEMBL_617637 (CHEMBL1101246) Displacement of biotinylated-VEGF-A125 from NRP1 b1 domain by cell-free assay
- ChEMBL_617638 (CHEMBL1101247) Displacement of [125I]VEGF-A from human NRP1 expressed in human A549 cells
- ChEMBL_617639 (CHEMBL1101248) Displacement of biotinylated-VEGF-A125 from human NRP1 expressed in human DU145 cells
- ChEMBL_881929 (CHEMBL2212532) Inhibition of HIF-1alpha-mediated VEGF secretion in human Hep3b cells by ELISA
- ChEMBL_1348204 (CHEMBL3270319) Inhibition of KDR in VEGF-stimulated HUVEC assessed as phosphorylation after 1 hr incubation
- ChEMBL_2525445 Inhibition of ERK1 phosphorylation in VEGF-stimulated serum-starved human HUVEC cells by immunoblotting analysis
- ChEMBL_2525446 Inhibition of ERK2 phosphorylation in VEGF-stimulated serum-starved human HUVEC cells by immunoblotting analysis
- ChEMBL_313055 (CHEMBL835746) Inhibition of vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cell proliferation
- ChEMBL_617636 (CHEMBL1101245) Displacement of [125I]VEGF-A from human NRP1 expressed in pig aortic endothelial cells
- ChEMBL_790781 (CHEMBL1926234) Inhibition of recombinant human VEGF-R2 using poly(Glu,Tyr)4:1 as substrate
- ChEMBL_1364556 (CHEMBL3295611) Inhibition of VEGFR-2 in HUVEC assessed as inhibition of VEGF (1 to 165)-induced cell proliferation preincubated for 2 hrs followed by VEGF (1 to 165) induction measured after 48 hrs by CCK8 assay
- ChEMBL_1339790 (CHEMBL3243495) Inhibition of VEGF-A production in human HCT116 cells after 12 hrs by alphaLISA assay
- ChEMBL_1555024 (CHEMBL3767602) Inhibition of VEGF induced VEGFR2 phosphorylation in HUVEC after 5 mins by Western blot analysis
- ChEMBL_2104429 (CHEMBL4812932) Inhibition of VEGFR2 in mouse NIH/3T3 cells assessed as reduction of VEGF-stimulated phosphorylation
- ChEMBL_462940 (CHEMBL929874) Inhibition of VEGF-stimulated VEGFR2 autophosphorylation in HUVEC cells at 50 nM relative to control
- ChEMBL_762507 (CHEMBL1816877) Inhibition of recombinant VEGF-R2 assessed as 33Pi incorporation after 60 mins by scintillation counting
- ChEMBL_942632 (CHEMBL2339394) Inhibition of recombinant VEGF-A/VEGFR-1 (unknown origin) interaction after 1 hr by ELISA
- ChEMBL_1795958 (CHEMBL4268075) Inhibition of biotinylated VEGF-A165 binding to rat NRP1/FC chimera after 40 mins by ELISA
- ChEMBL_1884097 (CHEMBL4385679) Displacement of biotinylated VEGF-A165 from human VEGFR-1 ECD incubated for 1 hr by ELISA
- ChEMBL_214499 (CHEMBL818398) Concentration required to achieve 50% inhibition of tyrosine phosphorylation on murine VEGF receptor (FLK-1 RTK).
- ChEMBL_434529 (CHEMBL914745) Inhibition of VEGF-induced phosphorylation of human KDR expressed in mouse NIH3T3 cell line by ELISA
- ChEMBL_881928 (CHEMBL2212531) Inhibition of HIF-1alpha-mediated VEGF expression in human Hep3b cells by luciferase reporter gene assay
- ChEMBL_1748926 (CHEMBL4183436) Inhibition of biotinylated VEGF-A binding to human NRP1 expressed in human DU145 cells after 2 hrs
- ChEMBL_2172376 (CHEMBL5057510) Inhibition of VEGFR2 phosphorylation in VEGF-induced HUVEC cells pretreated measured after overnight incubation by immunoblotting analysis
- ChEMBL_2292107 Inhibition of VEGF-induced human VEGFR2 autophosphorylation transfected in CHO cells measured after 2 hrs by chemiluminescence assay
- ChEMBL_2292136 Inhibition of VEGF-induced human VEGFR2 autophosphorylation expressed in HUVEC cells measured after 2 hrs by chemiluminescence assay
- ChEMBL_2496554 Inhibition of VEGF-induced human VEGFR2 autophosphorylation expressed in HUVEC cells measured after 2 hrs by chemiluminescence assay
- ChEMBL_434530 (CHEMBL914746) Inhibition of VEGF-induced phosphorylation of human KDR expressed in mouse NIH3T3 cell line by Western blot
- ChEMBL_565913 (CHEMBL960808) Inhibition of VEGF-induced phosphorylation of VEGFR2 by cell-based ELISA in presence of 8 uM ATP
- ChEMBL_793572 (CHEMBL1932340) Inhibition of VEGFR2 tyrosine kinase activity in VEGF-stimulated human U251 cells after 60 mins by ELISA
- ChEMBL_793573 (CHEMBL1932341) Inhibition of VEGFR1 tyrosine kinase activity in VEGF-stimulated human A498 cells after 60 mins by ELISA
- ChEMBL_540575 (CHEMBL1034027) Inhibition of VEGF-induced VEGFR2 phosphorylation expressed in CHO cells in presence of 8 uM ATP by ELISA
- ChEMBL_1462774 (CHEMBL3399576) Inhibition of HIF-1alpha in human Hep3B cells assessed as decrease in VEGF level after 20 hrs by ELISA
- ChEMBL_1927639 (CHEMBL4430711) Inhibition of biotinylated VEGF-A165 binding to recombinant NRP1 (unknown origin) measured after 2 hrs by competition binding assay
- ChEMBL_851754 (CHEMBL2156222) Activation of human HIF1alpha expressed in human U2OS cells assessed as increase in VEGF expression by RT-PCR analysis
- ChEMBL_2367960 Inhibition of [125I]-VEGF-A165 binding to NPR1 (unknown origin) transfected in HUVECs incubated for 2 hrs by plate reader analysis
- ChEMBL_1823987 (CHEMBL4323751) Inhibition of VEGFR2 in HUVEC assessed as inhibition of VEGF-induced tube formation incubated for 8 hrs by inverted microscopic method
- ChEMBL_1849682 (CHEMBL4350223) Inhibition of biotinylated human VEGF-A165 binding to recombinant human NRP1 receptor after 2 hrs by chemiluminescence analysis relative to control
- ChEMBL_2288784 Inhibition of DHODH in human HeLa cells assessed as reduction in hypoxia-induced VEGF expression incubated for 48 hrs by ELISA assay
- ChEMBL_2462411 Agonist activity at HIF-2alpha in human 786-0 cells assessed as increase in VEGF secretion incubated for 20 hrs by ELISA
- ChEMBL_2059413 (CHEMBL4714414) Inhibition of VEGFR2 in human U251 cells preincubated for 60 mins followed by VEGF stimulation and measured after 10 mins by ELISA
- ChEMBL_2317827 Inhibition of HIF-2alpha transcriptional activity in human 786-0 cells assessed as decrease in VEGF production incubated for 48 hrs by ELISA
- ChEMBL_1466620 (CHEMBL3404062) Inhibition of VEGFR2 in human U251 cells compound pretreated for 60 min before VEGF stimulation for 10 mins by phosphotyrosine ELISA cytoblot method
- ChEMBL_1518712 (CHEMBL3625035) Inhibition of HIF1 in human HEK293T cells transfected with pGL3-5HRE-VEGF and pRL-SV40 after 24 hrs by dual luciferase reporter assay
- ChEMBL_1645160 (CHEMBL3994089) Inhibition of HIF-1alpha transcriptional activity in human MCF7 cells assessed as reduction in VEGF secretion after 24 hrs under hypoxic condition by ELISA
- ChEMBL_808259 (CHEMBL1961229) Inhibition of human recombinant VEGF-R2 expressed in Sf9 cells using poly(E,Y)4:1 as substrate after 80 mins by scintillation counting
- ChEMBL_808260 (CHEMBL1961230) Inhibition of human recombinant VEGF-R3 expressed in Sf9 cells using poly(E,Y)4:1 as substrate after 80 mins by scintillation counting
- ChEMBL_810621 (CHEMBL2015672) Inhibition of human recombinant VEGF-R2 expressed in baculovirus expression system using GST-PLCgamma as substrate after 1 hr by time-resolved fluorescence analysis
- ChEMBL_1923480 (CHEMBL4426436) Inhibition of VEGFR2 (unknown origin) overexpressed in mouse NIH/ 3T3 cells incubated for 5 mins followed by stimulation with VEGF for 5 mins by immunoblotting analysis
- ChEMBL_2208073 (CHEMBL5121022) Inhibition of human VEGFR2 in HUVEC cells assessed as reduction in VEGFR2 phosphorylation incubated for 30 mins by anti-phospho VEGF Tyr 1175 antibody based assay
- ChEMBL_2339959 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 20 degreeC by isothermal titration calorimetry analysis
- ChEMBL_2339964 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 37 degreeC by isothermal titration calorimetry analysis
- ChEMBL_881913 (CHEMBL2212516) Inhibition of topoisomerase-1 in human U251 cells assessed as inhibition of HIF-1-mediated hypoxia-induced VEGF expression after 24 hrs by luciferase reporter gene assay
- ChEMBL_1748925 (CHEMBL4183435) Inhibition of biotinylated VEGF-A binding to N-terminal His6-tagged recombinant human NRP1-b1 domain expressed in Escherichia coli Rosetta Gami 2 (DE3) pLysS cells after 2 hrs
- ChEMBL_2339968 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 20 degreeC by direct titration based isothermal titration calorimetry analysis
- ChEMBL_2339969 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 37 degreeC by direct titration based isothermal titration calorimetry analysis
- ChEMBL_2339974 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 20 degreeC by reverse titration based isothermal titration calorimetry analysis
- ChEMBL_2339975 Binding affinity to human VEGF (13 to 107 residues) expressed in Escherichia coli Rosetta (DE3) cells assessed as dissociation constant at 37 degreeC by reverse titration based isothermal titration calorimetry analysis
- ChEMBL_841304 (CHEMBL2090045) Inhibition of FGFR1 in human SK-N-MC cells assessed as inhibition of FGF-induced ERK phosphorylation incubated for 1 hr prior to VEGF-stimulation measured after 30 mins by ELISA
- VEGF-R Kinase Inhibition Assay An ELISA assay was used to determine the ability of inhibitor to inhibit VEGF-R RTK activity. The compounds were incubated with enzyme 20 min at room temperature, with 2uM ATP in 96-well plates coated with a poly(Glu, Ala, Tyr) 6:3:1 random copolymer substrate. Phosphorylated tyrosine was then detected by sequential incubation with anti-phosphotyrosine antibody. IC50 data were interpolated by nonlinear regression using Microcal Origin software.
- VEGF-R Kinase Inhibition Assay An ELISA assay was used to determine the ability of inhibitor to inhibit VEGF-RRTK activity. The compounds were incubated with enzyme 20 min at room temperature, with 2uM ATP in 96-well plates coated with a poly(Glu, Ala, Tyr) 6:3:1 random copolymer substrate. Phosphorylated tyrosine was then detected by sequential in cubation with anti-phosphotyrosine antibody. IC50 data were interpolated by nonlinear regression using Microcal Origin software.
- VEGF-R2 Receptor-Linked Tyrosine Kinase Assay Compounds were evaluated for their ability to inhibit the kinase activity using an ELISA-based assay. The 96-well microtiter plate is coated with substrate solution. The reaction is initiated by adding recombinant human VEGF-R2 to reaction mixture containing inhibitor compound. After the incubation, the detection antibody, Eu-N1 antiphosphotyrosine (PT66) antibody, was added, and the fluorescence of the resulting solution was measured using the Victor Multilabel Counter.
- ELISA Assay VEGF: HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 μL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.). Data defined as % of DFO stimulation is used to calculate EC50 values with GraphPad Prism 4 software (San Diego, Calif.).
- VEGF ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 ceils per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 uL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Qpti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.). Data defined as % of DFO stimulation is used to calculate EC50 values with GraphPad Prism 4 software (San Diego, Calif.).
- VEGF ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 uL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.). Data defined as % of DFO stimulation is used to calculate EC50 values with GraphPad Prism 4 software (San Diego, Calif.).
- ChEMBL_1465276 (CHEMBL3406495) Inhibition of HIF1 in human HT1080 cells transfected with 5xHRE/pGL3/VEGF/E1b reporter plasmid pre-incubated for 1 hr followed by incubation under hypoxia conditions for 24 hrs by HRE-driven luciferase reporter gene assay
- ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 uL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.).
- ChEMBL_829292 (CHEMBL2060139) Inhibition of HIF1 transcriptional activity in human LN229 cells expressing VEGF-HRE-V6R and coexpressing luciferase, lac Z gene incubated for 1 hr in normoxia condition followed by 24 hrs in hypoxia condition by reporter gene assay
- ChEBML_1689672 Displacement of biotinylated VEGF-A (165 residues) from humanized recombinant C-terminal His-tagged VEGFR-1 extracellular domain expressed in baculovirus infected Sf21 cells preincubated for 1 hr followed by btVEGF-A165 addition measured after 2 hrs by ELISA
- VEGF ELISA Assay The ability of the disclosed compounds to inhibit HIF-2alpha was measured by determining VEGF expression in 786-O cells. About 7500 786-O cells were seeded into each well of a 96-well, white, clear bottom plate (07-200-566, Fisher Scientific) with 200 ul growth medium. Four hours later, compounds were dispensed into wells by Tecan D300e digital dispenser with starting concentration of 10 uM and log of dilution down to 1 nM as final concentration. Each concentration of treatment was performed in duplicate. About 20 hours later, medium was removed and fresh medium was added, followed by compounds treatment as described above. After 24 hours, cell culture medium was collected to determine VEGF concentration using an ELISA kit (R&D systems, cat #DVE00) following the manufacturer's instruction.
- ChEMBL_1977171 (CHEMBL4610306) Inhibition of recombinant human biotinylated VEGF-165A expressed in HEK293 cells binding to immobilized recombinant full length human FC-tagged NRP1 (chimera active) expressed in HEK293 cells using TMB as substrate incubated for 2 hrs followed by substrate addition by competitive ELISA
- Enzyme Assay The inhibition of ALK tyrosine kinase activity may be demonstrated using known methods, for example using the recombinant kinase domain of the ALK in analogy to the VEGF-R kinase assay described in J. Wood et al. Cancer Res. 60, 2178-2189 (2000).
- ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 μL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.).
- ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 μL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.).
- VEGF ELISA Assay The ability of the disclosed compounds to inhibit HIF-2α was measured by determining VEGF expression in 786-O cells. About 7500 786-O cells were seeded into each well of a 96-well, white, clear bottom plate (07-200-566, Fisher Scientific) with 200 ul growth medium. Four hours later, compounds were dispensed into wells by Tecan D300e digital dispenser with starting concentration of 10 uM and log of dilution down to 1 nM as final concentration. Each concentration of treatment was performed in duplicate. About 20 hours later, medium was removed and fresh medium was added, followed by compounds treatment as described above. 24 hours later, cell culture medium was collected to determine VEGF concentration using an ELISA kit (R&D systems, cat #DVE00) following the manufacturer's instruction.The EC50 is calculated by GraphPad Prism using the dose-response-inhibition (four parameter) equation. The plate with cells was then subjected to CellTiter-Glo luminescence cell viability assay (Promega) to determine the effect of these compounds on cell numbers after the above treatment.
- ELISA Assay HEK293 cells are seeded in 96-well poly-lysine coated plates at 20,000 cells per well in DMEM (10% FBS, 1% NEAA, 0.1% glutamine). Following overnight incubation, the cells are washed with 100 uL of Opti-MEM (Gibco, Carlsbad, Calif.) to remove serum. Compound in DMSO is serially diluted (beginning with 100 μM) in Opti-MEM and added to the cells. The conditioned media is analyzed for VEGF with a Quantikine human VEGF immunoassay kit (R&D Systems, Minneapolis, Minn.). Optical density measurements at 450 nm are recorded using the Spectra Max 250 (Molecular Devices, Sunnyvale, Calif.). Data defined as % of DFO stimulation is used to calculate EC50 values with GraphPad Prism 4 software (San Diego, Calif.).
- FLIPR Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 96-well fibronectin coated black-walled plates overnight at 37.degree. C./5% CO.sub.2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37.degree. C. Cells were washed 4 times (Original Cell Wash, Labsystems) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 .mu M) or at concentrations ranging from 0.01 to 10.0 .mu M followed by VEGF stimulation (5 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 96 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples. IC.sub.50 values for test compounds were calculated from % inhibition of VEGF stimulated responses in the absence of inhibitor.
- 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.
- FLIPR (Fluorometric Imaging Plate Reader) Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 96-well fibronectin coated black-walled plates overnight at 37 °C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37 °C. Cells were washed 4 times (Original Cell Wash, Labsystems) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 μM) or at concentrations ranging from 0.01 to 10.0 μM followed by VEGF stimulation (5 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 96 wells using a cooled CCD camera.
- Phospho ELISA Assay Adult human dermal lymphatic microvascular endothelial cells (HMVEC-dLyAD) (Cat# CC-2810, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in Endogro MV complete (Cat#SCME004, Millipore) at 200,000 cells/well (volume 1 mL), and the plates incubated at 37 C. and 5% CO2 for 6 hours. The media was replaced with Endogro Basal (Cat # SCME-BM, Millipore)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37 C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 uL of Total VEGFR2 capture antibody (Part #841888, Human Total VEGFR3/Flt4 ELISA Kit, Cat # DYC3491, R&D Systems), or Phospho VEGF R3Capture antibody (Part #841885, Human Phospho VEGF R3/Flt4 ELISA Kit, Cat# DYC2724, R&D Systems). The plates were covered and incubated at room temperature overnight. The coating antibody was flicked out and the plates washed three times with Wash Buffer.
- VEGFR2 Cellular Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight @ 37° C./5%CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples. IC50 values for test compounds were calculated from % inhibition of VEGF stimulated responses in the absence of inhibitor.
- VEGF-R Kinase Inhibition Assay Tha assay was using human VEGFR-2 in DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) format. IC50 is the concentration of inhibitor that inhibits 50% of kinase activity that catalyzes the transfer of the terminal phosphate to the the copolymer substrate. Compounds are tested using halflog dilutions from 10 through 0.0001 uM. IC50 values are calculated by least squares regression using the Hill equation for the best fit of all the points in the dose response curve.
- FLIPR (Fluorometric Imaging Plate Reader) Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight @37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (EIx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL).
- ELISA Assay VEGF: Four hours later, serial dilutions of 10x compound stocks were made in growth medium from 500xDMSO stocks, and 20 uL of those 10x stocks were added to each well to make final concentrations as follows (uL): 20, 6.67, 2.22, 0.74, 0.25, 0.082, 0.027, 0.009, 0.003, 0.001, and 0. Each concentration had duplicated wells. About 20 hours later, medium was removed by suction and each well was supplied with 180 uL of growth medium. About 20 uL freshly-made 10x compound stocks were added to each well. About 24 hours later, cell culture medium was removed for the determination of VEGFA concentration using an ELISA kit purchased from R&D systems by following the manufacturer's suggested method.
- VEGFR2 Cellular Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight @ 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 μM) or at concentrations ranging from 0.0001 to 10.0 μM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera.
- PDGFRbeta Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
- VEGFR2 Cellular Assay Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight @ 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
- In Vitro Receptor Tyrosine Kinase Assay These tests measure the ability of compounds to inhibit the enzymatic activity of recombinant human c-Met/HGF receptor and VEGF receptor enzymatic activity. A 1.3-kb cDNA corresponding to the intracellular domain of c-Met or c-Met IC (Genbank accession number NP000236-1 amino acid 1078 to 1337) is cloned into the BamHI/XhoI sites of the pBlueBacHis2A vector (Invitrogen) for the production of a histidine-tagged version of that enzyme. This construct is used to generate recombinant baculovirus using the Bac-N-Blue system according to the manufacturer's instructions (Invitrogen). The c-Met IC protein is expressed in Hi-5 cells (Trichoplusia Ni) upon infection with recombinant baculovirus construct. Briefly, Hi-5 cells grown in suspension and maintained in serum-free medium (Sf900 II supplemented with gentamycin) at a cell density of about 2x106 cells/ml are infected with the above-mentioned viruses at a multiplicity of infection (MOI) of 0.2 during 72 hours at 27 C.
- Kinase Assay VEGF receptor tyrosine kinase (90 ng/measuring point) was incubated for 10 min at 22 C. in the presence of different concentrations of test substances (0 uM, and within the range 0.001-10 M) in 30 ul of assay buffer [40 mM Tris/HCl pH 5.5; 10 mM MgCl2; 1 mM MnCl2; 3 uM Na ortho-vanadate; 1.0 mM dithiothreitol; 8 uM adenosine trisphosphate (ATP); 0.96 ug/measuring point poly-(Glu4Tyr); 0.2 uCi/measuring point 33P-gamma ATP; 1.4% dimethyl sulphoxide]. The reaction was stopped by adding EDTA solution (250 mM; pH 8.0; 15 ul/measuring point).From each reaction mixture, 15 ul were applied to P30 filter strips (Wallac), and unincorporated 33P-ATP was removed by washing the filter strips three times, for 10 min in each case, in 0.5% phosphoric acid. After drying the filter strips for 1 hour at 70 C., the filter strips were covered with scintillator strips (MeltiLex A, Wallac) and stoved for 1 hour at 90 C.
- VEGF-R2 Binding Assay A competition binding assay (DiscoveRx KINOMEscan) was used to measure the ability of the compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, et al, Nature Biotechnology (2005) 23, 329-336; Karaman, et al, Nature Biotechnology (2008) 26, 127-132).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32 ° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule.
- 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.
- ELISA Assay About 7500 of 786-O cells in 180 μL of growth medium were seeded into each well of a 96 well plate with white clear bottom on the first day (07-200-566, Fisher Scientific) in the layout presented in FIG. 9.Four hours later, serial dilutions of 10× compound stocks were made in growth medium from 500×DMSO stocks, and 20 μL of those 10× stocks were added to each well to make final concentrations as follows (μM): 20, 6.7, 2.2, 0.74, 0.25, 0.082, 0.027, 0.009, 0.003, 0.001, 0. Each concentration had duplicated wells. About twenty hours later, the growth medium was removed by suction and each well was supplied with 180 μL of growth medium. About 20 μl freshly-made 10× compound stocks were added to each well. About twenty four hours later, cell culture medium was removed for the determination of VEGF concentration using an ELISA kit purchased from R&D systems by following the manufacturer's suggested method. The EC50 was calculated by GraphPad Prism using the dose-response-inhibition (four parameter) equation. The cell seeded plate was then subjected to CellTiter-Glo luminescence cell viability assay (Promega) by adding about 50 μL of Celltiter Glo reagent into each well and shaking the plate for 8 minutes at 550 rpm (Thermomixer R, Eppendorf). The luminescence signals were read in a plate reader (3 second delay, 0.5 second/well integration time, Synergy 2 multi Detection Microplate reader) immediately.
- Kinase Assay The cytoplasmic domain of the human VEGF receptor (VEGFR-2) was expressed as a Histidine-tagged fusion protein following infection of insect cells using an His engineered baculovirus. His-VEGFR-2 was purified to homogeneity, as determined by SDS-PAGE, using nickel resin chromatography. Kinase assays were performed in 96 well microtiter plates that were coated overnight with 30 .mu g of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates were incubated with 1% BSA and then washed four times with PBS prior to starting the reaction. Reactions were carried out in 120 .mu L reaction volumes containing 3.6 .mu M ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl.sub.2, 0.1 mM MnCl.sub.2 and 0.2 mM Na.sub.3 VO.sub.4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 0.5 ng of purified protein. Following a ten minute incubation at 25.degree. C., the reactions were washed four times with PBS containing 0.05% Tween-20. 100 .mu l of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate was diluted 1:10000 in PBS-Tween-20 and added to the wells for 30 minutes. Following four washes with PBS-Tween-20, 100 .mu l of O-phenylenediamine Dihydrochloride in Phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 .mu l of 2.5N H.sub.2 SO.sub.4 to each well and read using a microplate ELISA reader set at 492 nm.
- VEGF-R2 Binding Assay A competition binding assay (DiscoveRx KINOMEscan ) was used to measure the ability of the compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, et al, Nature Biotechnology (2005) 23, 329-336; Karaman, et al, Nature Biotechnology (2008) 26, 127-132).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SeaBlock (Pierce), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% Tween 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SeaBlock, 0.17× phosphate buffered saline, 0.05% Tween 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% Tween 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% Tween 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and then diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.