- Carter, TA; Wodicka, LM; Shah, NP; Velasco, AM; Fabian, MA; Treiber, DK; Milanov, ZV; Atteridge, CE; Biggs, WH; Edeen, PT; Floyd, M; Ford, JM; Grotzfeld, RM; Herrgard, S; Insko, DE; Mehta, SA; Patel, HK; Pao, W; Sawyers, CL; Varmus, H; Zarrinkar, PP; Lockhart, DJ Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A 102: 11011-6 (2005)
- Traxler, PM; Wacker, O; Bach, HL; Geissler, JF; Kump, W; Meyer, T; Regenass, U; Roesel, JL; Lydon, N Sulfonylbenzoyl-nitrostyrenes: potential bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase. J Med Chem 34: 2328-37 (1991)
- Trinks, U; Buchdunger, E; Furet, P; Kump, W; Mett, H; Meyer, T; Müller, M; Regenass, U; Rihs, G; Lydon, N Dianilinophthalimides: potent and selective, ATP-competitive inhibitors of the EGF-receptor protein tyrosine kinase. J Med Chem 37: 1015-27 (1994)
- Million, ME; Boiziau, J; Parker, F; Tocque, B; Roques, BP; Garbay, C Inhibition of the EGF-stimulated cellular proliferation of ER 22 cells by hydroxybiphenyl derivatives. J Med Chem 38: 4693-703 (1995)
- Gazit, A; Osherov, N; Gilon, C; Levitzki, A Tyrphostins. 6. Dimeric benzylidenemalononitrile tyrophostins: potent inhibitors of EGF receptor tyrosine kinase in vitro. J Med Chem 39: 4905-11 (1996)
- Gazit, A; Chen, J; App, H; McMahon, G; Hirth, P; Chen, I; Levitzki, A Tyrphostins IV--highly potent inhibitors of EGF receptor kinase. Structure-activity relationship study of 4-anilidoquinazolines. Bioorg Med Chem 4: 1203-7 (1996)
- Traxler, PM; Furet, P; Mett, H; Buchdunger, E; Meyer, T; Lydon, N 4-(Phenylamino)pyrrolopyrimidines: potent and selective, ATP site directed inhibitors of the EGF-receptor protein tyrosine kinase. J Med Chem 39: 2285-92 (1996)
- 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)
- Gazit, A; Osherov, N; Posner, I; Yaish, P; Poradosu, E; Gilon, C; Levitzki, A Tyrphostins. 2. Heterocyclic and alpha-substituted benzylidenemalononitrile tyrphostins as potent inhibitors of EGF receptor and ErbB2/neu tyrosine kinases. J Med Chem 34: 1896-907 (1991)
- Traxler, P; Bold, G; Frei, J; Lang, M; Lydon, N; Mett, H; Buchdunger, E; Meyer, T; Mueller, M; Furet, P Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines. J Med Chem 40: 3601-16 (1997)
- Liu, T; Shirai, R; Matsui, T; Umezawa, K; Iwasaki, S Synthesis and biological activity of 5-[(2,5-dihydroxybenzyl)amino]salicylic acid analogs as inhibitors of EGF receptor-associated protein tyrosine kinase Bioorg Med Chem Lett 7: 365-368 (1997)
- Chen, H; Boiziau, J; Parker, F; Maroun, R; Tocque, B; Roques, BP; Garbay-Jaureguiberry, C Synthesis and structure-activity studies of a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity. J Med Chem 36: 4094-8 (1994)
- 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)
- Myers, MR; Setzer, NN; Spada, A; Zulli, AL; Hsu, CJ; Zilberstein, A; Johnson, SE; Hook, LE; Jacoski, MV The preparation and sar of 4-(anilino), 4-(phenoxy), and 4-(thiophenoxy)-quinazolines: Inhibitors of p56lck and EGF-R tyrosine kinase activity Bioorg Med Chem Lett 7: 417-420 (1997)
- Chen, H; Boiziau, J; Parker, F; Mailliet, P; Commerçon, A; Tocque, B; Le Pecq, JB; Roques, BP; Garbay, C Structure-activity relationships in a series of 5-[(2,5-dihydroxybenzyl)amino]salicylate inhibitors of EGF-receptor-associated tyrosine kinase: importance of additional hydrophobic aromatic interactions. J Med Chem 37: 845-59 (1994)
- Hennequin, LF; Ballard, P; Boyle, FT; Delouvrié, B; Ellston, RP; Halsall, CT; Harris, CS; Hudson, K; Kendrew, J; Pease, JE; Ross, HS; Smith, P; Vincent, JL Novel 4-anilinoquinazolines with C-6 carbon-linked side chains: synthesis and structure-activity relationship of a series of potent, orally active, EGF receptor tyrosine kinase inhibitors. Bioorg Med Chem Lett 16: 2672-6 (2006)
- Sun, L; Cui, J; Liang, C; Zhou, Y; Nematalla, A; Wang, X; Chen, H; Tang, C; Wei, J Rational design of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones as a novel class of inhibitors of epidermal growth factor receptor (EGF-R) and Her2(p185(erbB)) tyrosine kinases. Bioorg Med Chem Lett 12: 2153-7 (2002)
- Rewcastle, GW; Murray, DK; Elliott, WL; Fry, DW; Howard, CT; Nelson, JM; Roberts, BJ; Vincent, PW; Showalter, HD; Winters, RT; Denny, WA Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors. J Med Chem 41: 742-51 (1998)
- ChEMBL_66568 (CHEMBL679549) Inhibitory activity against recombinant tyrosine kinase EGF-R (EGF-R ICD)
- ChEMBL_473868 (CHEMBL936823) Inhibition of EGF
- ChEMBL_477145 (CHEMBL921756) Displacement of radiolabeled EGF from EGF receptor expressed in human MX1 cells
- ChEMBL_5000 (CHEMBL621147) Inhibition of EGF-stimulated tyrosine phosphorylation in A431 cells expressing EGF-R
- ChEMBL_5002 (CHEMBL621149) Inhibition of EGF-dependent autophosphorylation of EGF-R in human A431 cells
- ChEMBL_5167 (CHEMBL884002) Inhibition of EGF-induced Tyrosine phosphorylation in A431 cells expressing EGF-R
- ChEMBL_63603 (CHEMBL676147) Inhibition of EGF-dependent DNA synthesis in EGF-receptor expressing ER22 cells
- ChEMBL_65106 (CHEMBL674005) Inhibition of EGF-stimulated DNA synthesis in ER 22 (EGF-receptor expressing) cells
- ChEMBL_65107 (CHEMBL674006) Inhibition of EGF-stimulated DNA synthesis in ER 22 (EGF-receptor expressing) cells
- ChEMBL_2276618 Inhibition of EGF-R (unknown origin)
- ChEMBL_64435 (CHEMBL672267) Inhibition of EGF receptor kinase
- ChEBML_78739 Inhibition of polyGAT phosphorylation by EGF receptor
- ChEMBL_1363799 (CHEMBL3294216) Inhibition of EGF receptor (unknown origin)
- ChEBML_64439 Inhibition of epidermal growth factor receptor (EGF-R)
- ChEBML_64453 Inhibition of EGF-receptor autophosphorylation in intact cells
- ChEBML_67041 Inhibition of epidermal growth factor receptor (EGF-R)
- ChEBML_78512 Inhibition of EGF receptor overexpressing HN5 cell proliferation
- ChEMBL_63601 (CHEMBL675248) Inhibition of heparin-binding HB-EGF shedding
- ChEMBL_63789 (CHEMBL677393) Inhibitory concentration against EGF receptor by autophosphorylation
- ChEMBL_66719 (CHEMBL674642) Inhibition of EGF-R Tyrosine kinase (TK)
- ChEMBL_78739 (CHEMBL689288) Inhibition of polyGAT phosphorylation by EGF receptor
- ChEMBL_78740 (CHEMBL689289) Inhibition of polyGAT phosphorylation by EGF receptor
- ChEMBL_29499 (CHEMBL642000) Competitive inhibition of ATP binding to EGF-R
- ChEMBL_63788 (CHEMBL677392) Inhibitory concentration against EGF receptor by polyGAT phosphorylation
- ChEMBL_64439 (CHEMBL674014) Inhibition of epidermal growth factor receptor (EGF-R)
- ChEMBL_67041 (CHEMBL677879) Inhibition of epidermal growth factor receptor (EGF-R)
- ChEMBL_78512 (CHEMBL690548) Inhibition of EGF receptor overexpressing HN5 cell proliferation
- ChEMBL_103915 (CHEMBL711650) Inhibition of EGF-dependent mouse keratinocyte MK cell proliferation
- ChEMBL_305171 (CHEMBL832761) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate
- ChEMBL_306070 (CHEMBL833025) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate
- ChEMBL_306243 (CHEMBL831153) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate
- ChEMBL_354895 (CHEMBL853150) Antiproliferative activity against EGF-stimulated human KB cell line
- ChEMBL_1657723 (CHEMBL4007193) Inhibition of EGF-induced EGFR phosphorylation in human A431 cells preincubated for 60 mins followed by EGF induction measured after 10 mins by ELISA
- ChEMBL_1856530 (CHEMBL4357259) Inhibition of KRAS in human MIAPaca2 cells assessed as decrease in EGF-stimulated ERK1/2 phosphorylation preincubated for 2 hrs followed by EGF stimulation
- ChEMBL_1929276 (CHEMBL4432452) Inhibition of EGFR phosphorylation in EGF-stimulated human KB cells preincubated for 1 hr followed by EGF stimulation measured after 6 mins by ELISA
- ChEMBL_1291543 (CHEMBL3118715) Inhibition of EGF-stimulated autophosphorylation of EGFR in human KB cells incubated for 1 hr prior to EGF challenge measured after 6 mins by ELISA
- ChEMBL_1646279 (CHEMBL3995335) Inhibition of EGF induced EGFR phosphorylation in human KB cells preincubated for 90 mins followed by EGF addition for 5 mins by sandwich ELISA method
- ChEMBL_1674677 (CHEMBL4024820) Inhibition of EGF-stimulated wild type EGFR phosphorylation in human LoVo cells incubated for 2 hrs followed by EGF stimulation for 10 mins by ELISA
- ChEMBL_1778842 (CHEMBL4235834) Inhibition of EGF-induced EGFR activation in human A431 cells pretreated for 60 mins followed by EGF addition and measured after 10 mins by ELISA
- ChEMBL_2567999 Inhibition of EGFR phosphorylation in EGF-stimulated human NCI-H358 cells preincubated with compound for 2 hrs followed by EGF stimulation for 10 mins by ELISA
- ChEMBL_807903 (CHEMBL1959286) Inhibition of EGF-induced EGFR autophosphorylation in human A431 cells incubated for 60 mins prior to EGF-induction measured after 10 mins by phosphotyrosine ELISA
- ChEBML_64437 Tested for inhibition of EGF-receptor tyrosine kinase in intact cells
- ChEBML_67061 Inhibition of Epidermal growth factor receptor (EGF-R) tyrosine kinase activity
- ChEMBL_202292 (CHEMBL814059) Cytotoxic effect on EGF-receptor overexpressing HN5 squamous carcinoma cells
- ChEMBL_202293 (CHEMBL814060) Cytotoxic effect on EGF-receptor overexpressing HN5 squamous carcinoma cells
- ChEMBL_334486 (CHEMBL862515) Inhibition of EGF stimulated human erbB1 autophosphorylation in NIH3T3 cells
- ChEMBL_462993 (CHEMBL928914) Inhibition of EGF-stimulated human EGFR autophosphorylation in KB cells
- ChEMBL_605384 (CHEMBL1073378) Inhibition of wild type EGF-R by microplate scintillation counting
- ChEMBL_63592 (CHEMBL675239) Inhibition of heparin-binding epidermal growth factor (HB-EGF) shedding
- ChEMBL_64440 (CHEMBL674015) Tested in vitro for inhibition of EGF-receptor tyrosine kinase
- ChEMBL_66885 (CHEMBL676234) Cytotoxic effect on EGF-receptor overexpressing HN5 squamous carcinoma cells
- ChEMBL_93565 (CHEMBL705978) Inhibition of EGF-stimulated proliferation of KB cells in culture
- ChEMBL_1743840 (CHEMBL4178350) Inhibition of EGF-stimulated wild-type EGFR phosphorylation in human A549 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by sandwich ELISA
- ChEMBL_1892587 (CHEMBL4394508) Inhibition of EGFR in human A431 cells assessed as reduction in EGF-stimulated EGFR autophosphorylation preincuabted for 90 mins followed by EGF-stimulation by sandwich-ELISA
- ChEMBL_2546105 Inhibition of EGF-stimulated ERK5 autophosphorylation in human HeLa cells preincubated with compound for 1 hrs followed by EGF stimulation for 17 mins by SDS-PAGE analysis
- ChEMBL_2014665 (CHEMBL4668243) Inhibition of EGF-induced EGFR phosphorylation in human A431 cells preincubated for 24 hrs followed by EGF stimulation and measured after 10 mins by HTRF based immunoassay
- ChEMBL_311891 (CHEMBL833860) Inhibition of EGF-stimulated Elk1-luciferase reporter assay in HeLa cells
- ChEMBL_63613 (CHEMBL676157) Inhibition of epidermal growth factor receptor (EGF-R) in A431 cells.
- ChEMBL_64437 (CHEMBL674012) Tested for inhibition of EGF-receptor tyrosine kinase in intact cells
- ChEMBL_887629 (CHEMBL2217238) Inhibition of EGF-induced autophosphorylation of EGFR in human A431 cells
- ChEMBL_1613067 (CHEMBL3854867) Irreversible inhibition of full length human ERBB1 autophosphorylation transfected in EGF-stimulated mouse NIH/3T3 cells incubated for 2 hrs followed by stimulation with EGF for 10 mins
- ChEMBL_1659932 (CHEMBL4009544) Inhibition of EGF-induced wild-type EGFR phosphorylation in human A431 cells pre-incubated for 1 hr followed by EGF stimulation for 30 mins by Western blot analysis
- ChEMBL_2145651 (CHEMBL5029931) Inhibition of EGF-induced phosphorylation of wild type EGFR in human LoVo cells preincubated for 2 hrs followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_946792 (CHEMBL2344596) Inhibition of EGFR in human A431 cells assessed as inhibition of EGF-induced tyrosine phosphorylation incubated for 60 mins prior to EGF-activation measured 10 mins by ELISA
- ChEMBL_1649110 (CHEMBL3998244) Inhibition of EGFR in human A431 cells assessed as reduction in EGF induced phosphorylation preincubated for 60 mins followed by EGF addition measured after 10 mins by ELISA method
- ChEMBL_2145647 (CHEMBL5029927) Inhibition of EGF-induced phosphorylation of wild type EGFR in human NCI-H1975 cells preincubated for 120 mins followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_2145652 (CHEMBL5029932) Inhibition of EGF-induced phosphorylation of wild type EGFR in human A-431 cells preincubated for 2 hrs followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_2145653 (CHEMBL5029933) Inhibition of EGF-induced phosphorylation of wild type EGFR in human NCI-H2073 cells preincubated for 2 hrs followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_946709 (CHEMBL2343569) Inhibition of EGF-induced EGFR phosphorylation in human A431 cells overexpressing EGFR pretreated for 60 mins prior to EGF addition measured after 10 mins by phosphotyrosine ELISA cytoblot analysis
- ChEMBL_321388 (CHEMBL881766) In vitro inhibitory concentration EGF receptor with ATP concentration at 1/2Km
- ChEMBL_1832276 (CHEMBL4332284) Inhibition of EGF-induced EGFR phosphorylation at Tyr-residue in human HN5 cells preincubated for 24 hrs followed by EGF-stimulation and measured after 15 mins by Western blot analysis
- ChEMBL_1867398 (CHEMBL4368373) Inhibition of KRAS G12C mutant in human MIAPaCa2 cells assessed as reduction in EGF-induced ERK1/2 phosphorylation incubated for 4 hrs followed by EGF addition by luminescence based assay
- ChEMBL_1867404 (CHEMBL4368379) Inhibition of KRAS G12C mutant in human A549 cells assessed as reduction in EGF-induced ERK1/2 phosphorylation incubated for 4 hrs followed by EGF addition by luminescence based assay
- ChEMBL_1994717 (CHEMBL4628612) Inhibition of EGFR in human KB cells assessed as reduction in EGF-stimulated EGFR phosphorylation preincubated for 1 hr followed by EGF stimulation and measured after 6 mins by ELISA
- ChEMBL_63593 (CHEMBL675240) Inhibition of the heparin binding epidermal growth factor (HB-EGF) release from fibrosarcoma HT1080 transfectants expressing alkaline phosphate (AP) tagged HB-EGF stimulated by 12-O-tetradecanoylphorbol 13-acetate(TPA).
- ChEMBL_984001 (CHEMBL2434050) Inhibition of ADAM-17 in human A2780 cells assessed as inhibition of EGF-induced sALCAM release incubated for 30 mins prior to EGF induction measured after 18 hrs by ELISA
- ChEMBL_984004 (CHEMBL2434053) Inhibition of ADAM-17 in human A2774 cells assessed as inhibition of EGF-induced sALCAM release incubated for 30 mins prior to EGF induction measured after 18 hrs by ELISA
- ChEMBL_2145646 (CHEMBL5029926) Inhibition of EGF-induced phosphorylation of EGFR T790M/L858R double mutant in human NCI-H1975 cells preincubated for 120 mins followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_2227704 (CHEMBL5141217) Inhibition of EGF stimulated EGFR L858R/T790M double mutant phosphorylation in human NCI-H1975 cells preincubated for 4 to 5 hrs followed by EGF stimulation for 10 mins by AlphaLISA method
- ChEBML_63777 Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEBML_66903 Inhibition of Grb2 SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEBML_67053 Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_306418 (CHEMBL828089) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 1-10 nM
- ChEMBL_306444 (CHEMBL829088) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 10-50 nM
- ChEMBL_306445 (CHEMBL829089) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 25-50 nM
- ChEMBL_306459 (CHEMBL829536) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 6.7-20 nM
- ChEMBL_306537 (CHEMBL827826) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 10000-50000 nM
- ChEMBL_306538 (CHEMBL827827) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 3-5 nM
- ChEMBL_306555 (CHEMBL828300) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 1-10 nM
- ChEMBL_306556 (CHEMBL828301) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 4-10 nM
- ChEMBL_306557 (CHEMBL828302) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 5-25 nM
- ChEMBL_306573 (CHEMBL832921) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 50-250 nM
- ChEMBL_306574 (CHEMBL832922) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 6.7-20 nM
- ChEMBL_306626 (CHEMBL829928) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 1000-5000 nM
- ChEMBL_306642 (CHEMBL832979) Inhibition of EGF Receptor autophosphorylation in A431 cell lysate; Range = 1000-10000 nM
- ChEMBL_66569 (CHEMBL679550) Inhibition of EGF-stimulated autophosphorylation of Epidermal growth factor receptor in A431 cells
- ChEMBL_66570 (CHEMBL679551) Inhibition of EGF-stimulated autophosphorylation of epidermal growth factor receptor in A431 cells
- ChEMBL_1979829 (CHEMBL4612964) Inhibition of ERK5 in human HeLa cells assessed as reduction in EGF-induced ERK5 autophosphorylation pretreated for 1 hr followed by EGF stimulation and measured after 17 mins by 32p kinase assay
- ChEMBL_2227737 (CHEMBL5141250) Inhibition of EGF-stimulated EGFR ex19del (746 to 750) mutant phosphorylation in human PC-9 cells preincubated for 4 to 5 hrs followed by EGF stimulation for 10 mins by AlphaLISA method
- ChEMBL_152917 (CHEMBL758787) Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_221805 (CHEMBL843199) Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_2329793 Inhibition of ERK5 in human HeLa cells assessed as reduction in EGF-induced ERK5 autophosphorylation
- ChEMBL_63602 (CHEMBL676146) Inhibition of EGF-dependent DNA synthesis in ER 22 cells incubated with [3H]TdR
- ChEMBL_63776 (CHEMBL677218) Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_64442 (CHEMBL674017) Inhibition of EGF-stimulated autophosphorylation of epidermal growth factor receptor (EGFR) in A431 cells
- ChEMBL_64444 (CHEMBL674019) Inhibition of EGF-stimulated autophosphorylation of EGFR enzyme in A431 cells detected by immunoblotting
- ChEMBL_66901 (CHEMBL675383) Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_67046 (CHEMBL677884) Inhibition of autophosphorylation of epidermal growth factor receptor (EGF-R) in a cellular assay
- ChEMBL_719091 (CHEMBL1679302) Inhibition of EGF-induced BMK1 autophosphorylation in human HeLa cells by SDS-PAGE analysis
- ChEMBL_72184 (CHEMBL680002) Inhibition of Grb2-SH2 domain binding to phospho-EGF receptor intracellular C-terminal domain
- ChEMBL_1613066 (CHEMBL3854866) Irreversible inhibition of ERBB2 (unknown origin) autophosphorylation transfected in EGF-stimulated mouse T24 NIH/3T3 cells with extracellular binding domain of ERBB1 incubated for 2 hrs followed by stimulation with EGF for 10 mins
- ChEMBL_2134590 (CHEMBL4844200) Inhibition of KRas signaling in EGF-stimulated human NCI-H358 cells assessed as reduction in MEK phosphorylation preincubated for 4 hrs followed by EGF stimulation and measured after 10 mins by Western blot analysis
- ChEMBL_984003 (CHEMBL2434052) Inhibition of ADAM-17 in human SKOV3 cells transfected with luciferase gene assessed as inhibition of EGF-induced sALCAM release incubated for 30 mins prior to EGF induction measured after 18 hrs by ELISA
- ChEMBL_1451355 (CHEMBL3363374) Inhibition of EGFR extracellular/transmembrane domain-tagged Mer intracellular domain (unknown origin) expressed in 32D cells assessed as inhibition EGF-stimulated of phosphorylation pretreated for 1 hr prior to EGF stimulation by Western blot analysis
- ChEMBL_1451356 (CHEMBL3363375) Inhibition of EGFR extracellular/transmembrane domain-tagged Axl intracellular domain (unknown origin) expressed in 32D cells assessed as inhibition EGF-stimulated of phosphorylation pretreated for 1 hr prior to EGF stimulation by Western blot analysis
- ChEMBL_1451357 (CHEMBL3363376) Inhibition of EGFR extracellular/transmembrane domain-tagged Tyro3 intracellular domain (unknown origin) expressed in 32D cells assessed as inhibition EGF-stimulated of phosphorylation pretreated for 1 hr prior to EGF stimulation by Western blot analysis
- ChEMBL_1659933 (CHEMBL4009545) Inhibition of wild-type EGFR in human A431 cells assessed as reduction in EGF-induced Akt phosphorylation at S473 pre-incubated for 1 hr followed by EGF stimulation for 30 mins by Western blot analysis
- ChEMBL_76720 (CHEMBL688553) Inhibition of EGF-dependent proliferation of human and guinea pig keratinocytes; range 7-15 uM
- ChEMBL_793611 (CHEMBL1932379) Inhibition of EGFR autophosphorylation in EGF-stimulated human A431 cells after 1 hr by immunoblotting
- ChEMBL_2134588 (CHEMBL4844198) Inhibition of KRas signaling in EGF-stimulated human NCI-H358 cells assessed as reduction in Akt phosphorylation at ser473 residue preincubated for 4 hrs followed by EGF stimulation and measured after 10 mins by Western blot analysis
- ChEMBL_2134589 (CHEMBL4844199) Inhibition of KRas signaling in EGF-stimulated human NCI-H358 cells assessed as reduction in Akt phosphorylation at thr308 residue preincubated for 4 hrs followed by EGF stimulation and measured after 10 mins by Western blot analysis
- ChEMBL_1566936 (CHEMBL3790960) Inhibition of EGF-stimulated wild type EGFR autophosphorylation expressed in human A549 cells by sandwich ELISA
- ChEMBL_67040 (CHEMBL677878) Concentration required for inhibition of epidermal growth factor receptor (EGF-R) by tyrosine kinase enzyme activity
- ChEMBL_795532 (CHEMBL1935855) Inhibition of EGFR phosphorylation in EGF-stimulated human A431 after 2 hrs by Western blot analysis
- ChEMBL_624101 (CHEMBL1103432) Inhibition of ADAM17 in human A2774 cells assessed as inhibition of EGF-induced sALCAM release by ELISA
- ChEMBL_624104 (CHEMBL1103435) Inhibition of ADAM17 in human SKOV3 cells assessed as inhibition of EGF-induced sALCAM release by ELISA
- ChEMBL_624107 (CHEMBL1103438) Inhibition of ADAM17 in human MCF7 cells assessed as inhibition of EGF-induced sALCAM release by ELISA
- ChEMBL_63615 (CHEMBL676159) Concentration required to achieve 50% inhibition of tyrosine phosphorylation on human Epidermal growth factor receptor (EGF RTK).
- ChEMBL_793571 (CHEMBL1932339) Inhibition of EGFR tyrosine kinase activity in EGF-stimulated human A431 cells after 60 mins by ELISA
- ChEMBL_1622927 (CHEMBL3865279) Inhibition of human MerTK kinase domain (1585 to 3000 residues) expressed in HEK293 cells co-expressing rat EGFR LBD assessed as inhibition of EGF-stimulated MerTK phosphorylation preincubated for 30 mins followed by EGF stimulation for 15 mins by ELISA
- ChEMBL_2236515 (CHEMBL5150411) Inhibition of EGF stimulated EGFR autophosphorylation in human HN5 cells incubated for 24 hrs by Western blotting analysis
- ChEMBL_2525150 Inhibition of EGF-induced EGFR phosphorylation in human A-431 cells incubated for 3 hrs by Western blot analysis
- ChEMBL_695091 (CHEMBL1640839) Inhibition of EGF-induced EGFR phosphorylation in human CAL27 cells overexpressing EGFR after 16 hrs by Western blot
- ChEMBL_2222484 (CHEMBL5135818) Inhibition of EGF-stimulated wild-type EGFR phosphorylation in human A-431 cells incubated for 2 hrs by ELISA
- ChEMBL_624110 (CHEMBL1103441) Inhibition of ADAM17 in human MDA-MB-468 cells assessed as inhibition of EGF-induced sALCAM release by ELISA
- ChEMBL_65110 (CHEMBL674009) Inhibition of EGF-stimulated DNA synthesis in ER22 cells, by measuring [3H]Me-dT incorporation into ER 22 cells
- ChEMBL_2278659 Inhibition of EGR-induced EGFR phosphorylation in human A549 cells pretreated for 2 hrs followed by EGF stimulation by immune assay
- ChEMBL_65120 (CHEMBL678922) In vitro inhibition of EGF-stimulated DNA synthesis of ER 22 cells by following the incorporation of methyl-[3H]thymidine.
- ChEMBL_840627 (CHEMBL2090278) Inhibition of EGFR-mediated intracellular tyrosine phosphorylation in EGF-stimulated human A431 cells after 2.5 hrs by SDS-PAGE analysis
- ChEMBL_1973895 (CHEMBL4606713) Inhibition of recombinant human N-terminal GST-tagged EGFR (668 to end residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in EGF-stimulated EGFR phosphorylation preincubated for 2 hrs followed by EGF stimulation and measured after 30 mins by TMB peroxidase substrate based assay
- ChEMBL_2276372 Inhibition of human PCSK9 preincubated for 30 mins followed by EGF(A) addition and measured after 2 hrs by TR-FRET assay
- ChEMBL_2317271 Inhibition of Rac1 in human HeLa cells pretreated for 2 hrs followed by EGF stimulation for 2 mins by flow cytometric analysis
- ChEMBL_2317272 Inhibition of CDC42 in human HeLa cells pretreated for 2 hrs followed by EGF stimulation for 2 mins by flow cytometric analysis
- ChEMBL_2317273 Inhibition of CDC42 in human HeLa cells pretreated for 1 hr followed by EGF stimulation for 2 mins by flow cytometric analysis
- ChEMBL_2317274 Inhibition of Rac1 in human HeLa cells pretreated for 1 hr followed by EGF stimulation for 2 mins by flow cytometric analysis
- ChEMBL_2059412 (CHEMBL4714413) Inhibition of EGFR in human A431 cells preincubated for 60 mins followed by EGF stimulation and measured after 10 mins by ELISA
- ChEMBL_2075028 (CHEMBL4730562) Inhibition of wild type EGFR in human U87 cells assessed as heparin/human EGF stimulated phosphorylation after 1 hr by immunoblotting analysis
- ChEMBL_2146989 (CHEMBL5031335) Inhibition of EGR-induced EGFR phosphorylation in human A549 cells pretreated for 4 hr followed by EGF stimulation by Western blot analysis
- Kinase Assay Determination of EGF receptor kinase activity was performed as described using A431 membranes as the enzyme source and agiotensin II as substrate.
- Membrane Autophosphorylation Assay. The assay was using Swiss 3T3 cell membranes harboring EGF-R or PDGFR-beta. Receptor autophosphorylation was initiated by addition of [gamma-32P]ATP in the presence of EGF or PDGF. In order to test the effects of tyrphostins, these were added 15 min before addition of the growth factors. For quantification of radioactivity in the reaction, a PhosphorImager was used through electrophoresis gels. IC50 is the inhibitor concentration which inhibits 50% of EGF-R or PDGFR-beta kinase activity.
- ChEMBL_1466619 (CHEMBL3404061) Inhibition of EGFR in human A431 cells compound pretreated for 60 min before EGF stimulation for 10 mins by phosphotyrosine ELISA cytoblot method
- ChEMBL_1582062 (CHEMBL3817139) Inhibition of wild-type EGFR autophosphorylation in human LoVo cells incubated for 2 hrs followed by EGF-stiumlation for 10 mins by ELISA
- ChEMBL_1842127 (CHEMBL4342554) Inhibition of EGFR in human A431 cells incubated for 2 hrs followed by EGF stimulation and measured after 10 mins by immunoblot assay
- ChEMBL_2278677 Inhibition of EGR-induced wild type EGFR phosphorylation in human A-431 cells pretreated for 2 hrs followed by EGF stimulation by immune assay
- ChEMBL_763210 (CHEMBL1820121) Inhibition of EGFR-induced AP1 activation in human HeLa cells preincubated for 1 hr prior to EGF stimulation by luciferase reporter gene assay
- ChEMBL_763211 (CHEMBL1820122) Inhibition of EGFR-induced HIF1alpha activation in human HeLa cells preincubated for 1 hr prior to EGF stimulation by luciferase reporter gene assay
- ChEMBL_933730 (CHEMBL2320832) Inhibition of gamma secretase-mediated notch cleavage in HEK293 cells expressing human notch protein with truncated extracellular EGF repeat domain by sandwich immunoassay
- ChEMBL_1538409 (CHEMBL3737956) Inhibition of EGFR phosphorylation in human NCI-H1975 cells preincubated for 1 hr followed by stimulation with EGF for 8 mins by electrochemiluminescent immunoassay
- ChEMBL_1538410 (CHEMBL3737957) Inhibition of EGFR phosphorylation in human NCI-H292 cells preincubated for 1 hr followed by stimulation with EGF for 8 mins by electrochemiluminescent immunoassay
- ChEMBL_1567355 (CHEMBL3789495) Inhibition of wild type EGFR phosphorylation in human LoVo cells preincubated for 2 hrs followed by EGF stimulation measured after 30 mins by ELISA
- ChEMBL_1743852 (CHEMBL4178362) Inhibition of EGFR L858R mutant phosphorylation in human H3255 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by sandwich ELISA
- ChEMBL_1770089 (CHEMBL4222201) Inhibition of wild-type EGFR phosphorylation in human A431 cells preincubated for 1 hr followed by EGF stimulation measured after 45 mins by ELISA
- ChEMBL_2511030 Inhibition of EGFR-phosphorylation in EGFR expressing human A-431 cells incubated for 1 hr followed by EGF stimulation for 20 min by ELISA method
- ChEMBL_2511031 Inhibition of human HER2-phosphorylation in HER2 expressing mouse NIH3T3 cells incubated for 1 hr followed by EGF stimulation for 20 min by ELISA method
- ChEMBL_2511032 Inhibition of HER2-phosphorylation in HER2 expressing human BT-474 cells incubated for 1 hr followed by EGF stimulation for 20 min by ELISA method
- ChEMBL_2511033 Inhibition of HER2-phosphorylation in HER2 expressing human NCI-N87 cells incubated for 1 hr followed by EGF stimulation for 20 min by ELISA method
- ChEMBL_763212 (CHEMBL1820123) Inhibition of EGFR-induced Beta-casein activation in human HeLa cells preincubated for 1 hr prior to EGF stimulation by luciferase reporter gene assay
- ChEMBL_808129 (CHEMBL1961280) Inhibition of human recombinant EGF-R expressed in Sf9 cells using poly(E,Y)4:1 as substrate after 80 mins by scintillation counting
- EGFR assay The activity of EGFR, preactivated with EGF, is measured by its ability to transfer terminal phosphate from [gamma-32P]ATP to poly(GAT) substrate.
- ChEBML_1563976 Inhibition of EGFR T790M/L858R double mutant phosphorylation in human H1975 cells preincubated for 1 hr followed by EGF stimulation for 8 mins by electrochemiluminescent immunoassay
- ChEMBL_2432684 Inhibition of wildtype EGFR (unknown origin) phosphorylation transfected in human NCI-H2073 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by ELISA
- ChEMBL_1563976 (CHEMBL3784895) Inhibition of EGFR T790M/L858R double mutant phosphorylation in human H1975 cells preincubated for 1 hr followed by EGF stimulation for 8 mins by electrochemiluminescent immunoassay
- ChEMBL_1580544 (CHEMBL3813356) Inhibition of EGFR-TK in human A431 cell lysate assessed as reduction in EGF stimulated kinase activity after 60 mins using biotinylated peptide substrate by ELISA
- ChEMBL_1743841 (CHEMBL4178351) Inhibition of EGFR T790M/L858R double mutant phosphorylation in human H1975 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by sandwich ELISA
- ChEMBL_1743851 (CHEMBL4178361) Inhibition of EGFR exon 19 deletion mutant phosphorylation in human PC9 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by sandwich ELISA
- ChEMBL_2227705 (CHEMBL5141218) Inhibition of wild type EGFR in human A-431 cells preincubated for 4 to 5 hrs followed by EGF stimulation for 10 mins by AlphaLISA method
- ChEMBL_2432669 Inhibition of wildtype EGFR (unknown origin) phosphorylation transfected in human NCI-H2073 cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by HTRF assay
- ChEMBL_2475979 Binding affinity to sialyl LewisX mimetic 21a pretreated Blue-NHS-labelled E-selectin Lec-EGF/SCR1/SCR2 domain (unknown origin) measured after 20 mins by microscale thermophoresis
- ChEMBL_863014 (CHEMBL2175194) Inhibition of STAT1 dimerization in EGF-stimulated mouse NIH/3T3 cells overexpressing human EGFR assessed as suppression of STAT1-DNA interaction after 30 mins by EMSA
- ChEMBL_863016 (CHEMBL2175196) Inhibition of STAT3 dimerization in EGF-stimulated mouse NIH/3T3 cells overexpressing human EGFR assessed as suppression of STAT3-DNA interaction after 30 mins by EMSA
- ChEMBL_1838591 (CHEMBL4338724) Inhibition of EGFR phosphorylation at Y 1173 residues in human HeLa cells preincubated for 30 mins followed by EGF-stimulation and measured after 2 mins by ELISA
- ChEMBL_1933011 (CHEMBL4478663) Inhibition of AKT1 phosphorylation at Ser473 in EGF/insulin-stimulated human AN3CA cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933012 (CHEMBL4478664) Inhibition of AKT1 phosphorylation at Ser473 in EGF/insulin-stimulated human A2780 cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933013 (CHEMBL4478665) Inhibition of AKT1 phosphorylation at Thr308 in EGF/insulin-stimulated human AN3CA cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933014 (CHEMBL4478666) Inhibition of AKT1 phosphorylation at Thr308 in EGF/insulin-stimulated human A2780 cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_2549797 Inhibition of SOS1 in human HeLa cells assessed as reduction in RAS activation pretreated for 30 mins followed by EGF stimulation for 3 mins by G-LISA analysis
- ChEMBL_1743850 (CHEMBL4178360) Inhibition of EGFR T790M/exon 19 deletion mutant phosphorylation in human PC9-DRH cells preincubated for 2 hrs followed by EGF stimulation for 10 mins by sandwich ELISA
- ChEMBL_2549796 Inhibition of SOS1 in human Calu-1 cells assessed as reduction in RAS activation pretreated for 30 mins followed by EGF stimulation for 3 mins by G-LISA analysis
- ChEMBL_946791 (CHEMBL2344595) Inhibition of PDGFRbeta in human SF539 cells assessed as inhibition of PDGFR-BB-induced tyrosine phosphorylation incubated for 60 mins prior to EGF-activation measured 10 mins by ELISA
- ChEMBL_2222488 (CHEMBL5135822) Inhibition of EGF-stimulated EGFR phosphorylation in human A-431 cells incubated for 3 hrs followed by replacement with fresh medium without compound measured after 5 hrs by immunoblot analysis
- ChEMBL_1933015 (CHEMBL4478667) Inhibition of AKT1 in EGF/insulin-stimulated human AN3CA cells assessed as reduction in phosphorylation at Thr246 incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933016 (CHEMBL4478668) Inhibition of AKT1 in EGF/insulin-stimulated human A2780 cells assessed as reduction in phosphorylation at Thr246 incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1538415 (CHEMBL3737962) Inhibition of EGFR T790M/del (746 to 750) deletion mutant phosphorylation in erlotinib-resistant human PC9 cells preincubated for 1 hr followed by stimulation with EGF for 8 mins by electrochemiluminescent immunoassay
- ChEMBL_2293062 Inhibition of EGFR autophosphorylation in human NCI-H292 cells harboring harboring wild type EGFR preincubated for 60 mins followed by EGF stimulation and measured after 8 mins by SULFO-TAG based electrochemiluminescent assay
- ChEMBL_1912354 (CHEMBL4414937) Inhibition of EGFR in human A431 cells assessed as reduction in EGFR phosphorylation at Tyr1068 residues preincubated for 1 hr followed by human EGF stimulation and measured after 10 mins by HTRF assay
- ChEMBL_2526037 Inhibition of human erbB2 intracellular domain transfected in mouse NIH3T3 cells assessed as potency reduction in autophosphorylation of the chimera erbB2 incubated for 2 hrs in the presence of EGF stimulation by westernblot analysis
- ChEMBL_2291456 Inhibition of EGFR autophosphorylation at Tyr1068 residue in EGFR-amplified human A-431 cells preincubated with compound for 1 hrs followed by stimulation with human recombinant EGF and measured after 10 mins by HTRF-based analysis
- ChEMBL_2516787 Inhibition of EGFR in human A-431 cells membrane using peptide substrate pre incubated for 30 mins followed by ATP/peptide substrate addition and measured after 10 mins in presence of EGF by scintillation counting method
- ChEMBL_2516988 Inhibition of HER-1 in human A-431 cells assessed as inhibition of autophosphorylation of HER-1 incubated for 90 mins followed by EGF stimulation measured after 10 mins in presence of anti-EGFR Ab5 by ELISA analysis
- ChEMBL_2293065 Inhibition of EGFR L858R/T790M double mutant autophosphorylation in human NCI-H1975 cells harboring harboring EGFR L858R/T790M double mutant preincubated for 60 mins followed by EGF stimulation and measured after 8 mins by SULFO-TAG based electrochemiluminescent assay
- ChEMBL_2293063 Inhibition of EGFR del (746 to 750) mutant autophosphorylation in human PC-9 cells harboring harboring EGFR del (746 to 750) mutant preincubated for 60 mins followed by EGF stimulation and measured after 8 mins by SULFO-TAG based electrochemiluminescent assay
- ChEMBL_2293064 Inhibition of EGFR T790M/del (746 to 750 residues) mutant autophosphorylation in human PC-9/ER1 cells harboring harboring EGFR T790M/del (746 to 750 residues) mutant preincubated for 60 mins followed by EGF stimulation and measured after 8 mins by SULFO-TAG based electrochemiluminescent assay
- Autophosphorylation Assay IC50 is the inhibitor concentration which inhibits 50% of EGF-R or PDGFR-beta kinase autophosphorylation activity. The assay was performed in 96-well microtiter plates precoated with PDGFR-beta or EGFR-specific monoclonal antibodies to capture the respective receptors from lysates. The amount of phosphotyrosine present on the receptors was determined by incubating the immunolocalized receptor with a biotinylated monoclonal antibody directed against phosphotyrosine.
- Kinase Inhibition Assay The EGF-R kinase autophosphorylation activity was measured by DELFIA/time-resolved fluorometry with excitation at 340 nm and emission at 615 nm. Positive and negative controls were included in each plate by incubation of enzyme with or without ATP-MgCl2. The percentage of autophosphorylation inhibition by the compound was calculated using the equation: 100 - [(test - negative control)/(positive control - negativecontrol)]. The IC50 was obtained from curves of percentage inhibition.
- AlphaScreen Assay Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.).
- Activated ERK2 Kinase Assay Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R- [NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 μM ATP (all concentrations are final in the reaction) in a total volume of 10.25 μL. A 16-point, half-log dilution series of compounds at 41× final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 2-3 hours at ambient temperature. Reaction was terminated by addition of 10 μL of a 2× stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 μg/mL of AlphaScreen Protein A Acceptor Beads, 10 μg/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 μg/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively.
- ERK2 (20 pM) Kinase Assay Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
- Kinase Assay (New) Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
- In Vitro Kinase Assay In vitro kinase analysis was performed by using HTScan EGF Receptor Kinase Assay Kit (#7909) and HTScan HER2/ErbB2 Kinase Assay Kit (#7058) from Cell Signaling Technology Company. Operation steps refer to the specification of the used kits, and the method was used to measure the inhibition effects of the compound to be tested on substrate peptide phosphorylation of EGFR or Her2 receptor tyrosine kinase. At room temperature, ATP and substrate peptide as well as the compound to be tested were incubated in kinase reaction buffer, after a period of incubation, a stop buffer was added to terminate the reaction and the sample was transferred to a streptavidin-coated 96-well plate, the plate was washed and the phosphorylation level of substrate peptide was detected by using HRP-marked antibody against substrate phosphorylation, colorated with TMB, terminated the reaction with 2M sulfuric acid. Absorption at 450 nm wavelength was detected, and IC50 value (μM) was calculated.
- In vitro EGFR TK Inhibition Assays The experiments were performed according to the instructions of the manufacturer. The EGFR TK activity was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega). It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. Briefly, 96-well plates were precoated with EGFR, EGF, and tested compound in a ratio of 2:2:1 that were added.The reaction mixtures were incubated for 40 min at room temperature while being shaken. After completion of the reaction, 20 µL of ADP-GloTM Plus Luminescence kinase assay solution were added, incubate the plate for 30 min at room temperature. The corrected activity for each protein kinase target was determined by removing the blank control value. Luminescence signal was measured using aTecan Infinite Spectramax M2e microplate reader.
- Ba/F3 cell model generation and proliferation assays Ba/F3 cells were ordered from DSMZ (ACC300, Lot17) and grown in RPMI-1640 (ATCC 30-2001) + 10 % FCS + 10 ng/ml IL-3 at 37 °C in 5 % CO2 atmosphere. Plasmids containing EGFR mutants were obtained from GeneScript. To generate EGFR-dependent Ba/F3 models, Ba/F3 cells were transduced with retroviruses containing vectors that harbor EGFR isoforms. Platinum-E cells (Cell Biolabs) were used for retrovirus packaging. Retrovirus was added to Ba/F3 cells. To ensure infection, 4 μg/mL polybrene was added and cells were spinfected. Infection efficiency was confirmed by measuring GFP-positive cells using a cell analyzer. Cells with an infection efficiency of 10 % to 20 % were further cultivated and puromycin selection with 1 μg/mL was initiated. As a control, parental Ba/F3 cells were used to show selection status. Selection was considered successful when parental Ba/F3 cells cultures died. To evaluate the transforming potential of EGFR mutations, the growth medium was no longer supplemented with IL-3. Ba/F3 cells harboring the empty vector were used as a control. A switch from IL-3 to EGF was performed for Ba/F3 cells with the wildtype EGFR known for its dependency on EGF ligand. Approximately ten days before conducting the experiments, puromycin was left out. For proliferation assays (data in table 13), Ba/F3 cells were seeded into 96-well plates at 5 x 103 cells / 100 μL in growth media. Compounds were added by using a HP D3000 Digital Dispenser. All treatments were performed in technical triplicates. Treated cells were incubated for 72 h at 37 °C with 5 % CO2. CellTiter-Glo Luminescent Cell Viability Assay (Promega) was performed and chemoluminescence was measured by using the multilabel Plate Reader VICTOR X4. The raw data were imported into and analyzed with the Boehringer Ingelheim proprietary software MegaLab (curve fitting based on the program PRISM, GraphPad Inc.).
- Enzyme-linked immunosorbent assays (ELISA) Enzyme-linked immunosorbent assays (ELISA) were performed to measure phosphorylated EGFR levels using A431 cells (10% FBS). A431 (1.0*104 cells/40 μl/well) cells were seeded in 384 well. Compounds were dissolved in DMSO, serially diluted in DMSO and then were added, mixed, and incubated for 4 hours at 37° C., 5% CO2. Following the 4-hours incubation, cells were stimulated for 10 minutes with EGF (Invitrogen, cat #PHG0311) at a final concentration of 30 ng/mL in the incubator. The media was aspirated and cells were lysed in 10 lysis buffer with protease and phosphatase inhibitors (PerkinElmer, cat #ALSU-PEGFR-A50K). The plates were placed on a shaker for 5 minutes and then incubated for 30 min at 4° C. for complete lysis. The lysate was transferred to an Optiplate (Perkin Elmer, cat #6007290).Acceptor mix (PerkinElmer, cat #ALSU-PEGFR-A50K) was prepared just before use and 5 μL was dispensed to all the wells, followed by a 1.5-2 h incubation at room temperature in dark. The donor mix (PerkinElmer, cat #ALSU-PEGFR-A50K) was prepared under low light conditions prior to use and 511.1 of donor mix was added to all the wells under subdued lighting or green filters. The plates were placed on a shaker for 5 min, sealed, and incubated overnight at room temperature in dark. Plates were read on the Envision (PerkinElmer) using standard AlphaLISA settings.
- Enzyme-linked immunosorbent assays (ELISA) Table B4: Enzyme-linked immunosorbent assays (ELISA) were performed to measure phosphorylated EGFR levels using A431 cells (10% FBS). A431 (1.0*104cells/40 μl/well) cells were seeded in 384 well. Compounds were dissolved in DMSO, serially diluted in DMSO and then were added, mixed, and incubated for 4 hours at 37° C., 500 CO2. Following the 4-hours incubation, cells were stimulated for 10 minutes with EGF (Invitrogen, cat #PHG0311) at a final concentration of 30 ng/mL in the incubator. The media was aspirated and cells were lysed in 10 μL lysis buffer with protease and phosphatase inhibitors (PerkinElmer, cat #ALSU-PEGFR-A50K). The plates were placed on a shaker for 5 minutes and then incubated for 30 min at 4° C. for complete lysis. The lysate was transferred to an Optiplate (Perkin Elmer, cat #6007290). Acceptor mix (PerkinElmer, cat #ALSU-PEGFR-A50K) was prepared just before use and 5 μL was dispensed to all the wells, followed by a 1.5-2 h incubation at room temperature in dark. The donor mix (PerkinElmer, cat #ALSU-PEGFR-A50K) was prepared under low light conditions prior to use and 5 μl of donor mix was added to all the wells under subdued lighting or green filters. The plates were placed on a shaker for 5 min, sealed, and incubated overnight at room temperature in dark. Plates were read on the Envision (PerkinElmer) using standard AlphaLISA settings. The percentage of inhibition on EGFR phosphorylation was calculated following equation: % Inhibition=100×(LumHC−LumSample)/(LumHC−LumLC). The low and high controls (LC/HC) are generated from lysate from wells with cells treated with DMSO or 10 mM Staurosporine (BioAustralis, cat #BIA-S1086), respectively.
- HSD2 Activity Human descending colon epithelial stem cells were cultured as 3D organoids in accordance with Sato et al Gastroenterology. 2011 November; 141(5): 1762-72. Organoids were dissociated using TrypLE Express (life technologies) and plated on 96-well transwells (corning) in supplemented basal media (SBM-advanced DMEM/F12 containing 10 mM HEPES, 1:100 Glutamax, 1:100 penicillin/streptomycin, 1:100N2, 1:50 B27, 1 mM N-acetylcysteine, 10 nM [Leu15]-gastrin I) containing 100 ng/mL Wnt3A (W), 50 ng/mL EGF (E), 100 ng/mL Noggin (N), 500 ng/mL RSpondinl (R), 500 nM A83-01 (S) and 2.5 uM thiazovivin (T). Cultures were differentiated using SBM containing ENRA and 30 nM aldosterone on day 3, and cultures were used for assay on day 6 or 7. Compounds were diluted in DMSO and serial dilutions prepared by titrating in DMSO. Compounds were then diluted into DMEM/F12. Transwell plates containing descending colon cultures were washed twice with DMEM/F12 and compound was added to the apical compartment. Cells were incubated with test compound for 30 minutes at 37 C., 5% CO2 to equilibrate across the cell membrane. A second compound plate was prepared in which the serially diluted compounds in DMSO were diluted into DMEM/F12 containing 40 nM cortisol. Following the 30 minute pre-incubation step, the apical media was aspirated and compounds diluted in DMEM/F12 with 40 nM cortisol were added to the apical side of the transwell. The plate was then incubated for four hours at 37 C., 5% CO2. Cortisol levels were measured using a cortisol HTRF assay kit as described by the manufacturer (Cisbio). Concentration-response curves were then plotted and IC50 (and pIC50) values were determined using least squares non-linear regression.