BDBM23952 ESTRADIOL
BDBM25868 ESTRADIOL
ESTRADIOL BDBM22422
BDBM50270011 estradiol disulfate
Estradiol phosphate BDBM50333647 CHEMBL1642763
17beta-estradiol (E2) 17α-ethinylestradiol Ovocyclin US9561238, E2 [2,4,6,7-3H]-17beta-estradiol CHEMBL135 (1S,10R,11S,14S,15S)-15-methyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadeca-2,4,6-triene-5,14-diol US9034854, E2 CS336 [3H]]estradiol ESTRADIOL US9422324, E2 [2,4,6,7-3H]-E2 BDBM17292 US9040509, E2 [3H]-estradiol 17 beta-Estradiol Estradiol-17 alpha
17-ethinyl-3,17-estradiol Ethinylestradiol CHEMBL691 17alpha-ethynylestra-1,3,5(10)-triene-3,17beta-diol ETHINYL ESTRADIOL Ethynyl estradiol 17alpha-Ethinyl estradiol 17alpha-ethynylestradiol 17-ethinyl-3,17-oestradiol ethinyloestradiol 17-ethinylestradiol BDBM50187243
BDBM50016934 CHEBI:82520 ESTRADIOL MUSTARD
BDBM50423528 6-Dehydro-Estradiol CHEMBL223967
17beta-Estradiol-17-(beta-D-glucuronide) CHEMBL1697724 estradiol 17b glucuronide BDBM50344959 E17β-glucuronide
13-beta-hydroxyestradiol Estradiol BDBM50250613 CHEMBL499812
BDBM235683 US9688816, A Estradiol-17β (3)
BDBM50200937 CHEMBL219321 estradiol 3-O-sulfamate
BDBM50200938 CHEMBL220493 estradiol 17-O-sulfamate
BDBM50423526 CHEMBL121458 17Beta-Dihydroequillin 7-Dehydro-Estradiol
13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(17beta-estradiol) (estradiol)13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol([6,7-3H]-estradiol) 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(estradiol) CHEMBL135 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol(17alpha-estradiol) 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol 17beta-Estradiol 1,5-Dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one BDBM50005414 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol (estradiol) ESTRADIOL 15-methyltetracyclo[8.7.0.02,7.011,15]heptadeca-2,4,6-triene-5,14-diol Estradiol13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol 13-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol( Estradiol)
BDBM50309556 Per-6-(estradiol-17-ethynylbenzylamino)-6-deoxy-betacyclodextrin CHEMBL604989
CHEMBL78389 B-Homo-2-ethoxy-3,17beta-estradiol-7-tosylhydrazone BDBM50089345
2-methoxy-17beta-estradiol CHEMBL299613 BDBM50060957 Panzem 2-Hydroxyestradol 2-methyl ether
2-OH-estradiol 2-hydroxy-17beta-estradiol cid_247304 (17beta)-estra-1,3,5(10)-triene-2,3,17-triol US10864248, Compound 2 2-OH-E2 BDBM50262140 CHEMBL467987 estra-1,3,5(10)-triene-2,3,17beta-triol
BDBM50318292 CHEMBL1096975 17alpha-[2,2-Bis(ethoxycarbonyl)-1,3-dihydro-2H-inden-5-yl]-estradiol
CHEMBL1098710 BDBM50318293 17alpha-[2-Cyano-2-(ethoxycarbonyl)-1,3-dihydro-2H-inden-5-yl]-estradiol
CHEMBL605828 Per-6-(estradiol-17-(1H-1,2,3-triazol-4-yl))-6-deoxy-beta cyclodextrin BDBM50309553
Mono-6-(17-estradiol-17-(1H-1,2,3-triazol-4-yl))-6-deoxy-beta-cyclodextrin CHEMBL605714 BDBM50309552
BDBM20624 (1S,10R,11S,14R,15S)-15-methyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadeca-2(7),3,5-triene-5,14-diol 17alpha-estradiol
BDBM50159804 CHEMBL221802 estradiol 3,17-O,O-bis-sulfamate sulfamic acid (8R,9S,13S,14S,17S)-13-methyl-3-sulfamoyloxy-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl ester
cid_222757 MLS000028477 ESTRADIOL BENZOATE [(8R,9S,13S,14S,17S)-13-methyl-17-oxidanyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate BDBM56905 SMR000058343 benzoic acid [(8R,9S,13S,14S,17S)-17-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] ester [(8R,9S,13S,14S,17S)-17-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate
[(8R,9S,13S,14S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl] acetate acetic acid [(8R,9S,13S,14S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl] ester BDBM62878 MLS000069774 [(8R,9S,13S,14S,17S)-13-methyl-3-oxidanyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl] ethanoate cid_6852404 SMR000058696 17BETA-ESTRADIOL 17-ACETATE
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- ChEMBL_1358461 (CHEMBL3281629) Displacement of radiolabeled estradiol from estradiol receptor in rat uterine cytosol
- ChEMBL_515889 (CHEMBL993943) Displacement of [3H]estradiol from full-length human estrogen receptor beta by radiometric assay relative to estradiol
- ChEMBL_838505 (CHEMBL2078133) TP_TRANSPORTER: inhibition of estradiol-17beta-glucuronide uptake(estradiol-17beta-glucuronide:0.02uM) in OATP1B1-expressing HEK293 cells
- ChEMBL_66548 (CHEMBL677815) Inhibition of estradiol binding to estrogen receptor
- ChEMBL_879294 (CHEMBL2208692) Inhibition of human estradiol 17beta-dehydrogenase
- ChEBML_67517 Displacement of [3H]estradiol from Estrogen receptor alpha
- ChEBML_67826 Displacement of [3H]estradiol from Estrogen receptor beta
- ChEMBL_331401 (CHEMBL867043) Displacement of [3H]17beta-estradiol from ERalpha
- ChEMBL_331402 (CHEMBL867044) Displacement of [3H]17beta-estradiol from ERbeta
- ChEMBL_381820 (CHEMBL869166) Displacement of [3H]estradiol from human ERalpha
- ChEMBL_381821 (CHEMBL869167) Displacement of [3H]estradiol from human ERbeta
- ChEMBL_398763 (CHEMBL908035) Inhibition of estradiol to estrone conversion in MG63 cells
- ChEMBL_526667 (CHEMBL968170) Displacement of [3H]estradiol from estrogen receptor
- ChEMBL_815206 (CHEMBL2025223) Displacement of [3H]-17beta-estradiol from ERalpha
- ChEMBL_815207 (CHEMBL2025224) Displacement of [3H]-17beta-estradiol from ERbeta
- ChEBML_67369 Displacement of [3H]estradiol from human estrogen receptor alpha
- ChEMBL_207426 (CHEMBL813050) Inhibition of estradiol-stimulated T-47D cell proliferation.
- ChEMBL_2273897 Displacement of [3H]-17beta-estradiol to estrogen receptor (unknown origin)
- ChEMBL_438780 (CHEMBL889118) Displacement of [3H]estradiol from human recombinant ERalpha
- ChEMBL_438781 (CHEMBL889119) Displacement of [3H]estradiol from human recombinant ERbeta
- ChEMBL_504921 (CHEMBL946607) Displacement of [3H]estradiol from human recombinant ERalpha
- ChEMBL_509492 (CHEMBL1008128) Displacement of [3H]estradiol from human recombinant ERbeta
- ChEMBL_65909 (CHEMBL679353) Binding affinity for estrogen receptor, by competition with [3H]estradiol
- ChEMBL_65914 (CHEMBL677160) Displacement of [3H]-estradiol from estrogen receptor (ER)
- ChEMBL_67517 (CHEMBL872900) Displacement of [3H]estradiol from Estrogen receptor alpha
- ChEMBL_67826 (CHEMBL677222) Displacement of [3H]estradiol from Estrogen receptor beta
- ChEBML_67502 Inhibition of binding of 17 beta-estradiol to human Estrogen receptor alpha
- ChEBML_67827 Inhibition of binding of 17 beta-estradiol to human Estrogen receptor beta
- ChEMBL_172928 (CHEMBL779965) Displacement of [3H]estradiol from estrogen receptor in immature rat
- ChEMBL_1744049 (CHEMBL4178559) Displacement of estradiol from human ERalpha expressed in yeast cells
- ChEMBL_1744050 (CHEMBL4178560) Displacement of estradiol from human ERbetaa expressed in yeast cells
- ChEMBL_217747 (CHEMBL823810) Inhibition of estradiol-stimulated ZR-75-1-cell proliferation
- ChEMBL_217753 (CHEMBL824422) Inhibition of estradiol-stimulated ZR-75-1-cell proliferation
- ChEMBL_313633 (CHEMBL835777) pIC50 for 1 nM estradiol-induced Ishikawa cell proliferation
- ChEMBL_448344 (CHEMBL898603) Displacement of [3H]17beta-estradiol from recombinant human ERalpha
- ChEMBL_448345 (CHEMBL898604) Displacement of [3H]17beta-estradiol from recombinant human ERbeta
- ChEMBL_630247 (CHEMBL1106456) Displacement of [3H]estradiol from human placental 17beta-HSD2
- ChEMBL_66378 (CHEMBL673767) Displacement of [3H]estradiol from estrogen receptor in squirrel monkey
- ChEMBL_66403 (CHEMBL677097) Displacement of [3H]estradiol from estrogen receptor in immature rabbit
- ChEMBL_66409 (CHEMBL677103) Displacement of [3H]estradiol from estrogen receptor in immature rabbit
- ChEMBL_66410 (CHEMBL677104) Displacement of [3H]estradiol from estrogen receptor in immature rat
- ChEMBL_66411 (CHEMBL677105) Displacement of [3H]estradiol from estrogen receptor in mature rat
- ChEMBL_66539 (CHEMBL677806) Displacement of [3H]-estradiol from estrogen receptor in immature rat
- ChEMBL_66540 (CHEMBL677807) Displacement of [3H]-estradiol from estrogen receptor in mature rat
- ChEMBL_66852 (CHEMBL680737) Displacement of [3H]estradiol from estrogen receptor in mature rat
- ChEMBL_68148 (CHEMBL680724) Displacement of [3H]estradiol from estrogen receptor in squirrel monkey
- ChEBML_67812 Displacement of [3H]17-beta-estradiol from human Estrogen receptor beta
- ChEMBL_1282735 (CHEMBL3102032) Displacement of FITC-estradiol from human ERalpha by fluorescence polarization assay
- ChEMBL_1484215 (CHEMBL3537876) Inhibition of SULT1A1 in human MCF7 cells assessed as 17beta-estradiol sulfation
- ChEMBL_65915 (CHEMBL677161) Displacement of [3H]estradiol from Estrogen receptor in MCF-7 cells
- ChEMBL_66547 (CHEMBL677814) Displacement of [3H]- Estradiol from Estrogen receptor of rat uterine cytosol
- ChEMBL_66550 (CHEMBL677817) Displacement of [3H]- Estradiol from Estrogen receptor of rat uterine cytosol
- ChEMBL_66864 (CHEMBL675203) Irreversible inhibition of [3H]estradiol binding to lamb uterine estrogen receptor
- ChEMBL_66867 (CHEMBL675206) Displacement of [3H]-estradiol (E2) from sheep uterine estrogen receptor
- ChEMBL_67019 (CHEMBL677493) Displacement of [3H]estradiol from estrogen receptor-ligand binding domain
- ChEMBL_67037 (CHEMBL677875) Binding affinity for human estrogen receptor alpha by displacement of [3H]estradiol
- ChEMBL_67502 (CHEMBL679893) Inhibition of binding of 17 beta-estradiol to human Estrogen receptor alpha
- ChEMBL_67520 (CHEMBL682303) Inhibition of binding of 17 beta-estradiol to human Estrogen receptor alpha
- ChEMBL_67821 (CHEMBL677051) Inhibition of binding of 17 beta-estradiol to human Estrogen receptor beta
- ChEMBL_67827 (CHEMBL677223) Inhibition of binding of 17 beta-estradiol to human Estrogen receptor beta
- ChEMBL_808084 (CHEMBL1961192) Displacement of [3H]estradiol from rat uterine cytosolic estrogen receptor
- ChEMBL_99795 (CHEMBL874174) Inhibition of estradiol-stimulated MCF-7 breast adenocarcinoma cell proliferation
- ChEMBL_1577036 (CHEMBL3807012) Antagonist activity at ERalpha (unknown origin) transfected in 17beta-estradiol induced-HEK293 cells assessed as inhibition of estradiol-mediated protein transcriptional activity after 8 hrs by Luciferase reporter gene assay
- ChEBML_1581929 Displacement of [3H]-17beta-estradiol from human recombinant ERalpha expressed in rabbit reticulocytes
- ChEBML_1581930 Displacement of [3H]-17beta-estradiol from human recombinant ERbeta expressed in rabbit reticulocytes
- ChEBML_1664056 Displacement of [3H]estradiol from full-length human ERalpha receptor by scintillation counting
- ChEBML_67682 Binding affinity towards estrogen receptor beta by [3H]17-beta-estradiol displacement.
- ChEMBL_1770486 (CHEMBL4222598) Inhibition of UGT1A1 in human liver microsomes assessed as decrease in estradiol-3 glucuronidation
- ChEMBL_207428 (CHEMBL872717) Apparent binding affinity against estradiol-stimulated T-47D cell proliferation
- ChEMBL_305778 (CHEMBL827967) Inhibition of [3H]17-beta-estradiol binding to human estrogen receptor beta
- ChEMBL_305813 (CHEMBL829466) Inhibition of [3H]17-beta-estradiol binding to human estrogen receptor alpha
- ChEMBL_320908 (CHEMBL881235) Antagonistic activity against estrogen receptor beta in presence of 0.1 nM estradiol
- ChEMBL_320911 (CHEMBL881238) Antagonistic activity against estrogen receptor alpha in presence of 0.1 nM estradiol
- ChEMBL_468678 (CHEMBL951130) Displacement of [3H]estradiol from purified full length human ERalpha receptor
- ChEMBL_468679 (CHEMBL951131) Displacement of [3H]estradiol from purified full length human ERbeta receptor
- ChEMBL_552852 (CHEMBL958057) Inhibition of human placental 17beta-HSD2 using 17-beta-estradiol substrate
- ChEMBL_610117 (CHEMBL1074482) Displacement of [3H]estradiol from human recombinant ERalpha by liquid scintillation counting
- ChEMBL_618687 (CHEMBL1102465) Displacement of [3H]17beta-estradiol from human ERalpha expressed in SF9 cells
- ChEMBL_66542 (CHEMBL677809) In vitro displacement of [3H]estradiol from estrogen receptor of rat uterine cystol
- ChEMBL_66552 (CHEMBL677819) Equilibrium dissociation constant for rat uterine estrogen receptor binding [3H]estradiol
- ChEMBL_67038 (CHEMBL677876) Relative binding affinity for human estrogen receptor alpha compared to [3H]estradiol
- ChEMBL_67202 (CHEMBL678290) Relative binding affinity for human estrogen receptor beta compared to [3H]-estradiol
- ChEMBL_67488 (CHEMBL679325) Displacement of [3H]17-beta-estradiol from human Estrogen receptor alpha
- ChEMBL_67508 (CHEMBL682292) Displacement of [3H]17-beta-estradiol from human estrogen receptor alpha
- ChEMBL_68140 (CHEMBL680716) Displacement of [3H]17-beta-estradiol from MCF-7 cell lysate
- ChEMBL_826711 (CHEMBL2051158) Displacement of [3H]estradiol from ERalpha after 4 hrs by scintillation counting
- ChEMBL_826712 (CHEMBL2051159) Displacement of [3H]estradiol from ERbeta after 4 hrs by scintillation counting
- ChEMBL_877515 (CHEMBL2184039) Inhibition of 17beta-HSD1 assessed as conversion of estrone to estradiol by scintillation counting method
- ChEMBL_1476878 (CHEMBL3429912) Antagonist activity at ER alpha in human MCF7 cells assessed as inhibition of estradiol-induced PR expression treated for 24 hrs after incubation with estradiol for 30 mins by laser scanning imaging cytometer analysis
- ChEBML_67363 Displacement of [3H]17-beta-estradiol from full length human estrogen receptor alpha
- ChEBML_67680 Displacement of [3H]17-beta-estradiol from full length human estrogen receptor beta
- ChEMBL_100276 (CHEMBL712018) Inhibition of estradiol induced estrogen receptor transcriptional activation in MCF-7-2a cells
- ChEMBL_1664056 (CHEMBL4013737) Displacement of [3H]estradiol from full-length human ERalpha receptor by scintillation counting
- ChEMBL_217754 (CHEMBL881549) Apparent binding affinity against estradiol-stimulated ZR-75-1-cell proliferation
- ChEMBL_2280986 Displacement of [3H]-17beta-estradiol from bovine uterine estrogen receptor by competitive binding assay
- ChEMBL_306592 (CHEMBL832939) Inhibition of human estrogen receptor 2 using tritiated estradiol incubated for 3 hr
- ChEMBL_306604 (CHEMBL833494) Inhibition of human estrogen receptor 2 using tritiated estradiol incubated for 20 hr
- ChEMBL_326622 (CHEMBL863381) Displacement of [3H]17beta-estradiol from recombinant human ERalpha expressed in 293T cells
- ChEMBL_326623 (CHEMBL868723) Displacement of [3H]17beta-estradiol from recombinant human ERbeta expressed in 293T cells
- ChEMBL_536779 (CHEMBL984573) Displacement of [3H]estradiol from human estrogen receptor alpha/estrogen receptor beta
- ChEMBL_544137 (CHEMBL1014266) Inhibition of 17beta-HSD1 assessed as conversion of [14C]estradiol to [14C]estrone using NADP+
- ChEMBL_545688 (CHEMBL1034370) Displacement of fluorescein-labeled estradiol from human recombinant ERalpha by fluorescence polarization assay
- ChEMBL_545689 (CHEMBL1034371) Displacement of fluorescein-labeled estradiol from human recombinant ERbeta by fluorescence polarization assay
- ChEMBL_635744 (CHEMBL1120283) Displacement of [3H]17-beta-estradiol from human recombinant full length ERalpha
- ChEMBL_635745 (CHEMBL1120284) Displacement of [3H]17-beta-estradiol from human recombinant full length ERbeta
- ChEMBL_66071 (CHEMBL683101) Inhibition of estradiol binding to estrogen receptor in Human Breast cancer cytosol (3.3% ethanol)
- ChEMBL_66404 (CHEMBL677098) Displacement of [3H]17-beta-estradiol from Estrogen receptor of rabbit uterine tissue
- ChEMBL_66405 (CHEMBL677099) Displacement of [3H]17-beta-estradiol from Estrogen receptor of rabbit uterine tissue
- ChEMBL_67173 (CHEMBL681157) In vitro inhibition of [3H]17-beta-estradiol binding to human estrogen receptor alpha
- ChEMBL_67365 (CHEMBL676819) Binding affinity towards estrogen receptor alpha by [3H]17-beta-estradiol displacement.
- ChEMBL_67519 (CHEMBL682302) In vitro inhibition of [6,7-3H]-17beta-estradiol binding to human estrogen receptor alpha
- ChEMBL_67682 (CHEMBL682169) Binding affinity towards estrogen receptor beta by [3H]17-beta-estradiol displacement.
- ChEBML_67512 Ability to displace [3H]17-beta-estradiol from Estrogen receptor alpha by scintillation proximity assay.
- ChEBML_67823 Ability to displace [3H]17-beta-estradiol from Estrogen receptor beta by scintillation proximity assay.
- ChEMBL_100268 (CHEMBL712011) Agonist activity in transcriptional activation assay in MCF-7-2a cells compared to estradiol E2
- ChEMBL_101112 (CHEMBL710208) Inhibition of 17-beta-estradiol (10e-11 M) mediated MCF-7 cell proliferation
- ChEMBL_1487741 (CHEMBL3533735) Inhibition of OATP1B1 (unknown origin) expressed in HEK293 cells using estradiol-17beta-glucuronide substrate
- ChEMBL_1519264 (CHEMBL3624761) Antagonist activity at progesterone receptor in human MCF cells assessed as estradiol-induced receptor response
- ChEMBL_306408 (CHEMBL828749) Inhibition of [3H]17-beta-estradiol binding to rat ER beta expressed in Escherichia coli
- ChEMBL_306421 (CHEMBL828092) Inhibition of [3H]17-beta-estradiol binding to rat ER alpha expressed in Escherichia coli
- ChEMBL_306448 (CHEMBL829092) Inhibition of [3H]17-beta-estradiol binding to mouse ER beta expressed in Escherichia coli
- ChEMBL_306464 (CHEMBL829541) Inhibition of [3H]17-beta-estradiol binding to mouse ER alpha expressed in Escherichia coli
- ChEMBL_492828 (CHEMBL940317) Displacement of [3H]estradiol from ERalpha in rat uteri cytosol by competitive radiometric binding assay
- ChEMBL_514253 (CHEMBL975500) Inhibition of human placental 17beta-HSD1 assessed as conversion of [3H]estrone to [3H]17beta-estradiol
- ChEMBL_514254 (CHEMBL975501) Inhibition of human placental 17beta-HSD2 assessed as conversion of [3H]17beta-estradiol to [3H]estrone
- ChEMBL_535112 (CHEMBL983675) Displacement of [3H]estradiol from full length biotinylated human ERalpha by scintillation proximity assay
- ChEMBL_535113 (CHEMBL983676) Displacement of [3H]estradiol from full length biotinylated human ERbeta by scintillation proximity assay
- ChEMBL_552857 (CHEMBL958062) Inhibition of 17-beta-HSD1-mediated 17-beta-estradiol formation in human T47D cells
- ChEMBL_65908 (CHEMBL679352) Antiestrogenic activity in MCF-7-2a cells as concentration required to reduce estradiol effect by 50%
- ChEMBL_67363 (CHEMBL873603) Displacement of [3H]17-beta-estradiol from full length human estrogen receptor alpha
- ChEMBL_67680 (CHEMBL682167) Displacement of [3H]17-beta-estradiol from full length human estrogen receptor beta
- ChEMBL_815209 (CHEMBL2025226) Displacement of [3H]-estradiol from human full length recombinant ERalpha by scintillation proximity assay
- ChEMBL_815210 (CHEMBL2025227) Displacement of [3H]-estradiol from human full length recombinant ERbeta by scintillation proximity assay
- ChEMBL_88150 (CHEMBL693235) Stimulation of alkaline phosphatase activity in human endometrial Ishikawa cells with 1 nM E2 estradiol
- ChEMBL_88151 (CHEMBL693236) Stimulation of alkaline phosphatase activity in human endometrial Ishikawa cells with 1 nM E2 estradiol
- ChEBML_67500 In vitro concentration required to inhibit [3H]estradiol binding to human estrogen receptor alpha expressed in 293T cells
- ChEBML_67820 In vitro concentration required to inhibit [3H]estradiol binding to human estrogen receptor beta expressed in 293T cells
- ChEMBL_103299 (CHEMBL712253) Anti-estrogenicity for 50% inhibition of the MVLN cell luciferase activity due to 0.1 nM E2 (estradiol)
- ChEMBL_1436369 (CHEMBL3389572) Inhibition of OATP1B1 (unknown origin) expressed in HEK293 cells using [3H]estradiol-17beta-glucuronide substrate
- ChEMBL_1436370 (CHEMBL3389573) Inhibition of OATP1B3 (unknown origin) expressed in HEK293 cells using [3H]estradiol-17beta-glucuronide substrate
- ChEMBL_1590441 (CHEMBL3830811) Inhibition of human 17-beta-HSD14 using estradiol as substrate after 15 mins by fluorimetric assay
- ChEMBL_1717009 (CHEMBL4132009) Antagonist activity at ER in human MCF7 cells assessed as inhibition of 17beta-estradiol-induced cell proliferation
- ChEMBL_306229 (CHEMBL831137) Inhibition of [3H]17-beta-estradiol binding to human estrogen receptor beta expressed in Escherichia coli
- ChEMBL_306245 (CHEMBL831155) Inhibition of [3H]17-beta-estradiol binding to human estrogen receptor alpha expressed in Escherichia coli
- ChEMBL_312915 (CHEMBL826597) Antagonist effect against 10 pM 17-beta-estradiol induced MCF-7 cell proliferation
- ChEMBL_492829 (CHEMBL940318) Displacement of [3H]estradiol from ERbeta receptor in rat uteri cytosol by competitive radiometric binding assay
- ChEMBL_536773 (CHEMBL984567) Inhibition of estradiol-induced coactivator peptide SRC binding to recombinant estrogen receptor by fluorescence polarization assay
- ChEMBL_544140 (CHEMBL1013421) Inhibition of 17beta-HSD1 expressed in HEK 293 cells assessed as conversion of [14C]estrone to [14C]estradiol
- ChEMBL_67167 (CHEMBL681151) In vitro displacement of 0.5 nM [3H]17-beta-estradiol from human Estrogen receptor alpha
- ChEMBL_67329 (CHEMBL678663) In vitro displacement of 0.5 nM [3H]17-beta-estradiol from human Estrogen receptor beta
- ChEMBL_67341 (CHEMBL677746) In vitro inhibitory concentration against [3H]17-beta-estradiol binding to human estrogen receptor 2
- ChEMBL_751834 (CHEMBL1785589) Displacement of [3H]estradiol from human full-length ERbeta receptor by competitive radiometric binding assay
- ChEMBL_752205 (CHEMBL1786581) Inhibition of fluorescence-labeled 17beta-estradiol binding to ERalpha receptor after 2 hrs by fluorometric analysis
- ChEMBL_863171 (CHEMBL2175782) Displacement of [3H]-estradiol from human recombinant ERalpha expressed in Sf21 cells after 2 hrs
- ChEMBL_863172 (CHEMBL2175783) Displacement of [3H]-estradiol from human recombinant ERbeta expressed in Sf21 cells after 2 hrs
- Estrogen Receptor Binding Assay. The binding affinities were determined by a competitive radiometric binding assay using [3H]estradiol as tracer. The Kd for 3H-estradiol was determined by saturation binding to ER receptors. The IC50 values for compounds were converted to Ki using Cheng-Prusoff equation.
- ChEBML_68138 Tested by protection experiments to demonstrate the inactivation of estradiol dehydrogenase and the kinetic parameter Ki app was reported at a concentration of 20 uM
- ChEMBL_103159 (CHEMBL711304) Compound concentration required to induce transcriptional activation in MVLN cells comparable to 50% of 0.1 nM estradiol response
- ChEMBL_103160 (CHEMBL707870) Compound concentration required to induce transcriptional activation in MVLN cells equal to 50% of 0.1 nM estradiol response
- ChEMBL_1584556 (CHEMBL3820547) Inhibition of human placental microsomal aromatase using testosterone as substrate assessed as formation of estradiol after 10 mins
- ChEMBL_1649843 (CHEMBL3998977) Displacement of [3H]estradiol from full length recombinant human ESR1 expressed in insect cells by radiometric assay
- ChEMBL_1869284 (CHEMBL4370350) Antagonist activity at ERalpha in human MCF7 cells assessed as inhibition of estradiol-driven PR response by microplate cytometry
- ChEMBL_2193599 (CHEMBL5105959) Displacement of fluorescent labeled 17beta-estradiol from human ERbeta ligand binding domain by TR-FRET assay
- ChEMBL_320910 (CHEMBL881237) Binding affinity towards human estrogen receptor beta in a competitive binding assay using fluorescently labelled estradiol
- ChEMBL_320912 (CHEMBL881339) Binding affinity towards human estrogen receptor alpha in a competitive binding assay using fluorescently labelled estradiol
- ChEMBL_508157 (CHEMBL1008215) Inhibition of neutrophil elastase activation in beta-estradiol differentiated mouse EcoM-G cells after 24 hrs
- ChEMBL_508158 (CHEMBL1008216) Inhibition of cathepsin G activation in beta-estradiol differentiated mouse EcoM-G cells after 24 hrs
- ChEMBL_508159 (CHEMBL1008217) Inhibition of proteinase-3 activation in beta-estradiol differentiated mouse EcoM-G cells after 24 hrs
- ChEMBL_552334 (CHEMBL1006006) Inhibition of human MRP2-mediated estradiol-17-beta-glucuronide transport in Sf9 cells inverted membrane vesicles
- ChEMBL_555024 (CHEMBL957999) Inhibition of human recombinant 17beta-HSD1 expressed in HEK293 cell lysate assessed as conversion of radiolabeled estrone to estradiol
- ChEMBL_555026 (CHEMBL958789) Inhibition of human recombinant 17beta-HSD2 expressed in HEK293 cell lysate assessed as conversion of radiolabeled estrone to estradiol
- ChEMBL_555345 (CHEMBL954745) Inhibition of human recombinant 11beta-HSD1 expressed in HEK293 cell lysate assessed as conversion of radiolabeled estrone to estradiol
- ChEMBL_649075 (CHEMBL1218773) Displacement of [3H]estradiol from human ERalpha ligand binding domain expressed in Escherichia coli BL21 (DE3)
- ChEMBL_67203 (CHEMBL838887) In vitro binding affinity towards human Estrogen receptor 2 using [3H]17-beta-estradiol as radioligand
- ChEMBL_67518 (CHEMBL682301) In vitro binding affinity towards human Estrogen receptor alpha using [3H]17-beta-estradiol as radioligand
- ChEMBL_684637 (CHEMBL1285470) Inhibition of human placenta 17beta-HSD2 using [2,4,6,7-3H]-17beta-estradiol as substrate after 10 mins
- ChEMBL_1934443 (CHEMBL4480095) Inhibition of full length human ERalpha expressed in human HEC1 cells in presence of estradiol by luciferase reporter gene assay
- ChEMBL_2327364 Displacement of [3H]estradiol from human ER-alpha expressed in Sf21 cells incubated for 120 mins by scintillation counting analysis
- ChEMBL_313068 (CHEMBL835873) Antagonist activity as inhibition of 1 nM 17-beta-estradiol stimulated alkaline phosphatase induction in Ishikawa endometrial cells
- ChEMBL_448350 (CHEMBL898609) Antagonist activity at ERalpha expressed in yeast AH109 assessed as inhibition of 17-beta-estradiol induced alpha-galactosidase activity
- ChEMBL_454488 (CHEMBL886514) Displacement of [3H]estradiol from full length human recombinant ERalpha after 3 hrs by scintillation proximity assay
- ChEMBL_454489 (CHEMBL886515) Displacement of [3H]estradiol from full length human recombinant ERbeta after 3 hrs by scintillation proximity assay
- ChEMBL_544139 (CHEMBL1013420) Inhibition of 17beta-HSD1 expressed in HEK 293 cells assessed as conversion of [14C]estrone to [14C]estradiol using NADH
- ChEMBL_544141 (CHEMBL1013422) Inhibition of 17beta-HSD1 expressed in HEK 293 cells assessed as conversion of [14C]estrone to [14C]estradiol using NADPH
- Estrogen Receptor Binding Assay. Relative binding affinities were determined by a competitive radiometric binding assay using [3H]estradiol as tracer, and purified full-length human ER. Hydroxyapatite was used to adsorb the receptor-ligand complexes, and free ligand was washed away. The binding affinities are expressed as relative binding affinity (RBA) values with the RBA of estradiol set to 100%. The values given are the average +/- range or SD of two to three independent determinations. Estradiol binds to ERalpha with a Kd of 0.2 nM and to ERbeta with a Kd of 0.5 nM.
- ChEMBL_1345989 (CHEMBL3258023) Displacement of [2,4,6,7-3H]-estradiol from estrogen receptor in immature rat uterine cytosol by dextran-coated charcoal-adsorption assay
- ChEMBL_1363487 (CHEMBL3291709) Inhibition of human placental 17beta-HSD2 microsomal fraction using tritiated estradiol as substrate assessed as formation of estrone by HPLC analysis
- ChEMBL_1363489 (CHEMBL3291711) Inhibition of human placental 17beta-HSD1 cytosolic fraction using tritiated estrone as substrate assessed as formation of estradiol by HPLC analysis
- ChEMBL_1363494 (CHEMBL3291985) Inhibition of mouse liver 17beta-HSD2 microsomal fraction using tritiated estradiol as substrate assessed as formation of estrone by HPLC analysis
- ChEMBL_1484212 (CHEMBL3537873) Inhibition of human SULT1A1 expressed in Escherichia coli assessed as 17beta-estradiol sulfation at 100 nM by Michaelis-Menten equation analysis
- ChEMBL_1486476 (CHEMBL3531466) Inhibition of human recombinant UGT1A1 expressed in HEK293 cells assessed as reduction in estradiol 3-glucuronidation by LC-MS/MS method
- ChEMBL_1490069 (CHEMBL3536010) Inhibition of OATP1B3-mediated [3H]estradiol 17beta-glucuronide uptake in human OATP1B3 expressing HEK293/PDZK1 cells by scintillation counting
- ChEMBL_1717005 (CHEMBL4132005) Displacement of [3H]17beta-estradiol from ER in human MCF7 cells after 18 hrs by microbeta scintillation counting method
- ChEMBL_2149318 (CHEMBL5033716) Displacement of fluorescent estradiol from full-length ERalpha (unknown origin) measured after 2 hrs by fluorescence polarization assay
- ChEMBL_2228974 (CHEMBL5142487) Inhibition of UGT1A1 in human liver microsomes using beta-estradiol as substrate incubated for 40 mins by LC-MS/MS analysis
- ChEMBL_2249199 (CHEMBL5163409) Antagonist activity at ERalpha expressed in HEK293/Gal4 cells incubated for 24 hrs in presence of estradiol by luciferase reporter gene assay
- ChEMBL_2316391 Antagonist activity against ERalpha in estradiol stimulated HEK293T cells measuring luciferase activity after 24 hrs by luciferase reporter assay
- ChEMBL_2316395 Antagonist activity against ERbeta in estradiol stimulated HEK293T cells measuring luciferase activity after 24 hrs by luciferase reporter assay
- ChEMBL_492826 (CHEMBL940315) Displacement of fluorescein labeled estradiol from human recombinant ERalpha expressed in baculovirus infected insect cells by fluorescence polarization assay
- ChEMBL_492827 (CHEMBL940316) Displacement of fluorescein labeled estradiol from human recombinant ERbeta expressed in baculovirus infected insect cells by fluorescence polarization assay
- ChEMBL_647642 (CHEMBL1219993) Displacement of [2,4,6,7-3H]estradiol from human ERalpha expressed in HeLa cells after 18 hrs by liquid scintillation counting
- ChEMBL_67194 (CHEMBL678129) In vitro inhibition of 1 nM 17-beta-estradiol induced transcriptional activation in T47D cells expressing estrogen receptor beta
- ChEMBL_67501 (CHEMBL679892) In vitro inhibition of transcriptional activation induced by 1 nM 17-beta estradiol in T47D cells expressing estrogen receptor alpha
- ChEMBL_67516 (CHEMBL682300) Binding affinity for Estrogen receptor alpha of human breast cancer cells (MCF-7) by displacement of [3H]17-alpha-estradiol
- ChEMBL_841251 (CHEMBL2089654) Displacement of fluorescent labelled PGC-1alpha from estradiol-ERalpha complex after 2 to 4 hrs by TR-FRET assay
- ChEMBL_841252 (CHEMBL2089655) Displacement of fluorescent labelled PGC-1alpha from estradiol-ERbeta complex after 2 to 4 hrs by TR-FRET assay
- Estrogen Receptor Binding Assay and Ishikawa Assay The binding affinities were determined by a competitive radiometric binding assay using [3H]estradiol as tracer. The Kd for 3H-estradiol was determined by saturation binding to ER receptors. The IC50 values for compounds were converted to Ki using Cheng-Prusoff equation. Estrogenic stimulation and antagonism were measured in Ishikawa human endometrial tumor cells by alkaline phosphatase quantitation. The data were fitted to a linear interpolation to derive IC50 values for the antagonist mode and a percentage efficacy was calculated that blocks the 17beta-estradiol (1nM) stimulus.
- ChEMBL_1484074 (CHEMBL3537109) Inhibition of human OATP1B1 expressed in CHO cells using [3H]estradiol 17beta-D-glucuronide as substrate by liquid scintillation counting analysis
- ChEMBL_1580951 (CHEMBL3812619) Positive allosteric modulation of human FSHR expressed in rat granulocytes assessed as FSH-induced estradiol production after 72 hrs by radioimmunossay
- ChEMBL_306447 (CHEMBL829091) Inhibitory concentration against human ER beta expressed in Escherichia coli was determined using [3H]17-beta-estradiol as radio ligand
- ChEMBL_306463 (CHEMBL829540) Inhibitory concentration against human ER alpha expressed in Escherichia coli was determined using [3H]17-beta-estradiol as radio ligand
- ChEMBL_527767 (CHEMBL978703) Agonist activity at human ERalpha expressed in african green monkey CV1 cells by luciferase reporter gene assay relative to 17beta estradiol
- ChEMBL_619853 (CHEMBL1106730) Inhibition of human 17beta-HSD7 expressed in HEK293 cells assessed as inhibition of reduction of [14C]estrone into [14C]estradiol after 7 hrs
- ChEMBL_66406 (CHEMBL677100) Inhibitory activity against Estrogen receptor by displacement of [3H]17-beta-estradiol in ER-rich cytosol from rabbit uterine tissue
- ChEMBL_99805 (CHEMBL710408) In vitro antagonist effect on estrogen receptor alpha transcriptional activation in MCF-7 cells against 10 pM 17-beta-estradiol
- Radioligand Displacement Assay Binding affinity was measured by a scintillation proximity binding assay using [3H]4-OHT (for ERRgamma) or [3H]estradiol (for ERalpha) as radioligand.
- ChEMBL_1287642 (CHEMBL3110898) Mixed-type inhibition of ER-alpha (unknown origin) expressed in ER-negative human SKBR3 cells coexpressing ERE using estradiol as substrate
- ChEMBL_1491711 (CHEMBL3536903) Inhibition of human MRP2-mediated estradiol-17beta-D-glucuronide formation using inside-out membrane vesicles by LC-MS/MS analysis
- ChEMBL_1822722 (CHEMBL4322486) Displacement of [3H]-estradiol from human recombinant estrogen receptor expressed in insect cells incubated for 17 hrs by radioactive receptor binding assay
- ChEMBL_775706 (CHEMBL1912091) Transactivation activity of glucocorticoid receptor in HFF assessed as induction of aromatase activity by measuring beta-estradiol activity after 18 to 24 hrs by ELISA
- ChEBML_1685129 Antagonist activity at ERalpha (unknown origin) expressed in HEK293T cells assessed as inhibition of estradiol-induced response after 24 hrs by luciferase reporter gene assay
- ChEMBL_1361782 (CHEMBL3292467) Antagonist activity at estrogen receptor in human MCF7 cells assessed as inhibition of estradiol-induced cell proliferation after 5 days by WST-8 assay
- ChEMBL_1494637 (CHEMBL3529887) Inhibition of OATP1B1 (unknown origin) expressed in HEK293 cells assessed as reduction of [3H]estradiol-17beta-glucuronide uptake after 3 mins by beta-counting
- ChEMBL_1494638 (CHEMBL3529888) Inhibition of OATP1B3 (unknown origin) expressed in HEK293 cells assessed as reduction of [3H]estradiol-17beta-glucuronide uptake after 3 mins by beta-counting
- ChEMBL_1562015 (CHEMBL3779189) Inhibition of CDK8 (unknown origin) expressed in 7dF3 clone of human HEK293 cells preincubated for 2 hrs followed by addition of beta-estradiol by luciferase assay
- ChEMBL_1664063 (CHEMBL4013744) Downregulation of ERalpha in human MCF-7 cells assessed as reduction of estradiol-induced GREB1 mRNA levels after 24 hrs by RT-PCR method
- ChEMBL_1664064 (CHEMBL4013745) Downregulation of ERalpha in human MCF-7 cells assessed as reduction of estradiol-induced PgR mRNA levels after 24 hrs by RT-PCR method
- ChEMBL_1664065 (CHEMBL4013746) Downregulation of ERalpha in human MCF-7 cells assessed as reduction of estradiol-induced pS2 mRNA levels after 24 hrs by RT-PCR method
- ChEMBL_1744042 (CHEMBL4178552) Inhibition of human 17beta-HSD2 expressed in HEK293 cell lysates incubated for 10 mins using [2,4,6,7-3H]-estradiol and NAD+ by scintillation counting method
- ChEMBL_1767338 (CHEMBL4219450) Displacement of fluorescein-labeled estradiol (fluoromone) from human recombinant full-length estrogen receptor alpha after 2 hrs by fluorescence polarization assay
- ChEMBL_1767339 (CHEMBL4219451) Displacement of fluorescein-labeled estradiol (fluoromone) from human recombinant full-length estrogen receptor beta after 2 hrs by fluorescence polarization assay
- ChEMBL_822871 (CHEMBL2037903) Antagonist activity at Estrogen receptor expressed in human Ishikawa cells assessed as inhibition of estradiol-induced increase in alkaline phosphatase after 3 days by spectrophotometry
- ChEMBL_879539 (CHEMBL2208948) Inhibition of 17beta-HSD1 in human T47D cells assessed as decrease in transformation of [14C]estrone to [14C]-estradiol after 24 hrs by thin layer chromatography
- Competitive Radiometric Binding Assay Binding affinities were also determined by a competitive radiometric binding assay using 2 nM [3H]estradiol as tracer (PerkinElmer, Waltham, MA) and full-length purified human ERα (Pan Vera/Invitrogen, Carlsbad, CA), as reported previously. The RBA values were calculated using the following equation: IC50 estradiol/IC50 compound×100.
- ChEBML_1685205 Inhibition of recombinant human 17beta-HSD2 expressed in HEK293 cells using estradiol as substrate after 10 mins in presence of radiolabeled tracer substrate by scintillation counting method
- ChEMBL_1287641 (CHEMBL3110897) Competitive inhibition of ER-alpha (unknown origin) ligand binding pocket expressed in ER-negative human SKBR3 cells coexpressing ERE using estradiol as substrate
- ChEMBL_1337645 (CHEMBL3242833) Antagonist activity at human Gal4-fused ER-alpha expressed in HEK293 cells assessed as inhibition of 17beta-estradiol-induced effect by luciferase reporter gene assay
- ChEMBL_1337649 (CHEMBL3242837) Antagonist activity at human Gal4-fused ER-beta expressed in HEK293 cells assessed as inhibition of 17beta-estradiol-induced effect by luciferase reporter gene assay
- ChEMBL_1367283 (CHEMBL3300204) Inhibition of human placental 17beta-HSD2 microsomal fraction using [2, 4, 6, 7-3H]-estradiol as substrate after 20 mins by HPLC analysis
- ChEMBL_1684871 (CHEMBL4035350) Inhibition of human CYP1B1 expressed in Escherichia coli DH5alpha coexpressing human NADPH-P450 reductase using 4-estradiol as substrate in presence of NADP+ by HPLC analysis
- ChEMBL_1767356 (CHEMBL4219468) Displacement of fluorescein-labeled estradiol (fluoromone) from human recombinant full-length untagged estrogen receptor alpha expressed in Spodoptera frugiperda by fluorescence polarization assay
- ChEMBL_1767357 (CHEMBL4219469) Displacement of fluorescein-labeled estradiol (fluoromone) from human recombinant full-length untagged estrogen receptor beta expressed in insect cells by fluorescence polarization assay
- ChEMBL_1869281 (CHEMBL4370347) Antagonist activity at ERalpha in human MCF7 cells assessed as inhibition of estradiol-driven cell growth measured after 7 days by SYTOX green-based assay
- ChEMBL_1934447 (CHEMBL4480099) Displacement of fluorescein-labeled PGC1alpha peptide from human GST-tagged ERalpha LBD after 2 hrs in presence of estradiol-beta by TR-FRET assay
- ChEMBL_2246242 (CHEMBL5160452) Inhibition of STS in human MCF7 cells assessed as reduction in [3H]estradiol and [3H]estrone formation using [3H]estrone sulfate as substrate incubated for 20 hrs
- ChEMBL_2249242 (CHEMBL5163452) Antagonist activity at ERalpha Y537S mutant degradation in human SK-BR-3 cells incubated for 24 hrs in presence of beta-estradiol by luciferase gene assay
- ChEMBL_2249243 (CHEMBL5163453) Antagonist activity at ERalpha D538G mutant degradation in human SK-BR-3 cells incubated for 24 hrs in presence of beta-estradiol by luciferase gene assay
- ChEMBL_474434 (CHEMBL935185) Antagonist activity at human ER ligand binding domain expressed in african green monkey COS7 cells in presence of 17-beta-estradiol by Gal4 hybrid assay
- ChEMBL_527053 (CHEMBL980129) Antiestrogenic activity at estrogen receptor alpha expressed in yeast cells assessed as inhibition of 17-beta-estradiol-induced beta-galactosidase activity by two hybrid assay
- ChEMBL_622102 (CHEMBL1114101) Antagonist activity at estrogen receptor in human Ishikawa cells assessed as 17-beta-estradiol-induced alkaline phosphatase activity after 3 days by chemiluminescence assay
- ChEMBL_861318 (CHEMBL2173706) Inhibition of human placenta 17beta-HSD2 microsomal fraction using unlabelled and [2,4,6,7-3H]estradiol as substrate after 20 mins by HPLC analysis in presence of NAD+
- ChEMBL_988475 (CHEMBL2438124) Inhibition of human placenta 17beta-HSD2 microsomal fraction using unlabeled, [2,4,6,7-3H]-estradiol as substrate after 20 mins by HPLC analysis in presence of NAD+
- ChEMBL_1361049 (CHEMBL3295681) Antagonist activity at ER in human MCF7:WS8 cells assessed as inhibition of estradiol-induced cell growth by measuring DNA level after 7 days by fluorescence analysis
- ChEMBL_1649840 (CHEMBL3998974) Antagonist activity at ERalpha in tamoxifen-sensitive human MCF7:WS8 cells assessed as inhibition of estradiol-induced response after 18 hrs by luciferase reporter gene assay
- ChEMBL_1870077 (CHEMBL4371244) Antagonist activity at estrogen receptor beta (unknown origin) assessed as inhibition of estradiol-induced response after 22 hrs by cell-based luciferase reporter gene assay
- ChEMBL_1870096 (CHEMBL4371263) Antagonist activity at estrogen receptor alpha (unknown origin) assessed as inhibition of estradiol-induced response after 22 hrs by cell-based luciferase reporter gene assay
- ChEMBL_2046646 (CHEMBL4701345) Inhibition of estrogen sulfotransferase in human liver cytosol incubated for 30 mins in presence of [3H]-estradiol and adenosine -3'-phosphate 5'-phosphosulfate by liquid scintillation counting method
- ChEMBL_2198168 (CHEMBL5110684) Inhibition of estrone sulfatase in human MCF7 cells assessed as inhibition of [3H]estrone and [3H]estradiol formation using [3H]estrone sulfate as substrate incubated for 20 hrs
- ChEMBL_622103 (CHEMBL1114102) Antagonist activity at estrogen receptor in human MCF7 cells assessed as 17-beta-estradiol-induced cell proliferation after 24 hrs by [14C]thymidine incorporation assay
- ChEMBL_651562 (CHEMBL1227814) Antagonist activity at human wild type ERbeta expressed in HEK293T cells co-expressing ERE assessed as inhibition of estradiol-induced transactivation by luciferase reporter gene assay
- ChEMBL_651565 (CHEMBL1227817) Antagonist activity at human wild type ERalpha expressed in HEK293T cells co-expressing ERE assessed as inhibition of estradiol-induced transactivation by luciferase reporter gene assay
- ChEMBL_718372 (CHEMBL1679904) Antagonist activity at human ERalpha LBD in human MCF7 cells assessed as inhibition of estradiol-induced cell proliferation after up to 6 days by celltiter-glo assay
- ChEMBL_1343606 (CHEMBL3254234) Apparent inhibition of human placental microsomal aromatase assessed as [C14]estrone/[C14]estradiol formation using 0.075 to 0.515 uM [C14]androstenedione substrate by liquid scintillation counting
- ChEMBL_1484075 (CHEMBL3537110) Inhibition of human OATP1B3 expressed in BacMam baculovirus virus infected HEK MSR2 cells using [3H]estradiol 17beta-D-glucuronide as substrate by liquid scintillation counting analysis
- ChEMBL_1684870 (CHEMBL4035349) Inhibition of human CYP1B1 expressed in Escherichia coli DH5alpha coexpressing human NADPH-P450 reductase using 4-estradiol as substrate in presence of NADP+ by Michaelis-Menten plot analysis
- ChEMBL_1890699 (CHEMBL4392453) Inhibition of recombinant rat UGT1A1 using beta-estradiol as substrate preincubated for 5 mins followed by substrate addition and measured after 40 mins by LC-MS/MS analysis
- ChEMBL_2074716 (CHEMBL4730250) Antagonist activity at full length human ERalpha receptor assessed as inhibition of estradiol-induced activation incubated for 22 to 24 hrs by cell based luciferase reporter gene assay
- ChEMBL_2074717 (CHEMBL4730251) Antagonist activity at full length human ERbeta receptor assessed as inhibition of estradiol-induced activation incubated for 22 to 24 hrs by cell based luciferase reporter gene assay
- ChEMBL_2222785 (CHEMBL5136119) Displacement of [3H]-estradiol from recombinant full-length human estrogen beta receptor expressed in baculovirus infected insect cells incubated overnight by liquid scintillation counting analysis
- Estrogen Receptor Binding and Transcription Activation Assay Relative binding affinities were determined by a competitive radiometric binding assay using [3H]estradiol as tracer, and purified full-length human ER. Hydroxyapatite was used to adsorb the receptor-ligand complexes, and free ligand was washed away. The binding affinities are expressed as relative binding affinity (RBA) values with the RBA of estradiol set to 100%. The values given are the average +/- range or SD of two to three independent determinations. Estradiol binds to ERalpha with a Kd of 0.2 nM and to ERbeta with a Kd of 0.5 nM. EC50/IC50 values were obtained from transcription activation by ER gene transfection in human endometrial cancer cells.
- ChEMBL_101082 (CHEMBL708494) Antagonism of estrogen action in a mammary tumor cell line was assayed via inhibition of MCF-7 cell proliferation stimulated by 10 e-11 M 17-beta-estradiol (in vitro)
- ChEMBL_1890700 (CHEMBL4392454) Inhibition of UGT1A1 in human liver microsomes using beta-estradiol as substrate preincubated for 5 mins followed by substrate addition and measured after 40 mins by LC-MS/MS analysis
- ChEMBL_1890701 (CHEMBL4392455) Inhibition of UGT1A1 in rat liver microsomes using beta-estradiol as substrate preincubated for 5 mins followed by substrate addition and measured after 40 mins by LC-MS/MS analysis
- ChEMBL_536775 (CHEMBL984569) Inhibition of estradiol-induced coactivator peptide SRC1 binding to estrogen receptor alpha expressed in human HEC1 cells after 24 hrs by luciferase based mammalian two-hybrid assay
- ChEMBL_1339080 (CHEMBL3243177) Antagonist activity at ERbeta (unknown origin) expressed in human HepG2 cells assessed as inhibition of 17beta-estradiol-induced transcriptional activation after 24 hrs by ERE-luciferase reporter gene assay
- ChEMBL_1934446 (CHEMBL4480098) Displacement of europium-labeled SRC1 peptide from full length human recombinant ERalpha expressed in baculovirus expression system after 1.5 hrs in presence of estradiol-beta by TR-FRET assay
- ChEMBL_536774 (CHEMBL984568) Antagonist activity at estrogen receptor alpha expressed in human HEC1 cells assessed as inhibition of 1 nM estradiol-induced transcriptional activity after 24 hrs by luciferase reporter gene assay
- ChEMBL_536780 (CHEMBL984574) Antagonist activity at estrogen receptor alpha expressed in human HEC1 cells assessed as inhibition of 100 nM estradiol-induced transcriptional activity after 24 hrs by luciferase reporter gene assay
- ChEMBL_969612 (CHEMBL2406604) Antagonist activity at Gal4 DBD-fused human ERalpha LBD expressed in HEK293T cells assessed as inhibition of estradiol-induced transcriptional activation after 20 hrs by luciferase reporter gene assay
- ChEMBL_1670787 (CHEMBL4020675) Binding affinity to recombinant human GFP-fused ERalpha LBD (301 to 553 residues) expressed in Escherichia coli BL21(DE3) after 40 mins in presence of 17beta-estradiol by fluorescence polarization assay
- ChEMBL_1670788 (CHEMBL4020676) Binding affinity to recombinant human GFP-fused ERbeta LBD (259 to 498 residues) expressed in Escherichia coli BL21(DE3) after 40 mins in presence of 17beta-estradiol by fluorescence polarization assay
- ChEMBL_1674627 (CHEMBL4024656) Induction of selective estrogen receptor alpha degradation in human MCF7 cells harboring TK-ERE-Luc assessed as reduction in estradiol-induced GREB1 mRNA expression after 24 hrs by TaqMan assay
- ChEMBL_1674615 (CHEMBL4024644) Induction of selective estrogen receptor alpha degradation in human MCF7 cells harboring TK-ERE-Luc assessed as reduction in estradiol-induced transcriptional activity after 24 hrs by luciferase reporter gene assay
- ChEMBL_1678645 (CHEMBL4028922) Inhibition of human MRP2 overexpressed in Sf9 cell membrane vesicles assessed as uptake of [3H]-estradiol-17beta-D-glucuronide in presence of ATP and GSH measured after 20 mins by membrane vesicle transport assay
- ChEMBL_1678647 (CHEMBL4028924) Inhibition of human MRP4 overexpressed in Sf9 cell membrane vesicles assessed as uptake of [3H]-estradiol-17beta-D-glucuronide in presence of ATP and GSH measured after 20 mins by membrane vesicle transport assay
- ChEMBL_1778275 (CHEMBL4235267) Antagonist activity at recombinant human ERalpha expressed in HEK293 cells assessed as inhibition of estradiol-induced YFP-fused SRC1 coactivator recruitment measured after 1 hr in presence of Coelenterazine H by BRET assay
- ChEMBL_1993610 (CHEMBL4627505) Displacement of [3H]17-beta estradiol from GST-fused human recombinant ERalpha ligand binding domain expressed in Escherichia coli BL21alpha cells incubated for 1 hr by liquid scintillation counting method
- ChEMBL_1993611 (CHEMBL4627506) Displacement of [3H]17-beta estradiol from GST-fused human recombinant ERbeta ligand binding domain expressed in Escherichia coli BL21alpha cells incubated for 1 hr by liquid scintillation counting method
- ChEMBL_1678646 (CHEMBL4028923) Inhibition of human MRP3 overexpressed in Sf9 insect cell membrane vesicles assessed as uptake of [3H]-estradiol-17beta-D-glucuronide in presence of ATP and GSH measured after 10 mins by membrane vesicle transport assay
- ChEMBL_1776993 (CHEMBL4233985) Antagonist activity at full length recombinant human ERalpha expressed in baculovirus expression system assessed as inhibition of estradiol-induced Euphorium-labelled LTERHKILHRLLQEGSPSD peptide recruitment after 1.5 hrs by time-resolved fluorescence assay
- ChEMBL_1776994 (CHEMBL4233986) Antagonist activity at full length recombinant human ERbeta expressed in baculovirus expression system assessed as inhibition of estradiol-induced Euphorium-labelled QAQQKSLLQQLLTE peptide recruitment after 1.5 hrs by time-resolved fluorescence assay
- ChEMBL_1859158 (CHEMBL4360014) Antagonist activity at human full-length ERalpha expressed in non-human mammalian expression system assessed as inhibition of 17beta-estradiol-induced response measured after 22 to 24 hrs by luciferase reporter gene assay
- ChEMBL_1859159 (CHEMBL4360015) Antagonist activity at human full-length ERbeta expressed in non-human mammalian expression system assessed as inhibition of 17beta-estradiol-induced response measured after 22 to 24 hrs by luciferase reporter gene assay
- ChEMBL_2369988 Inhibition of recombinant human full-length C-terminal His-tagged HSD17B13 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- ChEMBL_2369989 Inhibition of recombinant human full-length C-terminal His-tagged HSD17B11 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- ChEMBL_2369992 Inhibition of recombinant mouse full-length C-terminal His-tagged HSD17B13 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- ChEMBL_616793 (CHEMBL1103117) Antagonist activity at human recombinant ERalpha LBD expressed in HEK293 cells assessed as inhibition of 0.3 nM estradiol-induced transcriptional activation after 16 to 18 hrs by GAL4-dependent luciferase reporter gene assay
- ChEMBL_616795 (CHEMBL1103119) Antagonist activity at human recombinant ERbeta LBD expressed in HEK293 cells assessed as inhibition of 0.3 nM estradiol-induced transcriptional activation after 16 to 18 hrs by GAL4-dependent luciferase reporter gene assay
- ChEMBL_786678 (CHEMBL1921531) Inhibition of human placental 17beta-HSD2 assessed as formation of [2,4,6,7-3H]-estrone using [2,4,6,7-3H]-estradiol as substrate after 20 mins by radio flow detector-based HPLC analysis in presence of NAD+ as cofactor
- ChEMBL_1484073 (CHEMBL3537108) Inhibition of human MRP2 expressed in baculovirus-infected Sf9 cell membrane vesicle using [3H]estradiol 17beta-D-glucuronide as substrate preincubated with vesicles for 5 mins prior to substrate addition by liquid scintillation counting analysis
- ChEMBL_1668112 (CHEMBL4018000) Antagonist activity at estrogen receptor in mouse GT1-7 cells harboring beta-galactosidase reporter gene assessed as reduction in 17beta-estradiol induced response measured after 20 to 24 hrs by luciferase reporter gene assay
- ChEMBL_1750176 (CHEMBL4184936) Antagonist activity at CMV-fused ERalpha-LBD-GAL4-DBD (unknown origin) expressed in HEK293T cells assessed as inhibition of estradiol-induced receptor transactivation after 16 hrs by bright-Glo luciferase reporter gene assay
- ChEMBL_1750177 (CHEMBL4184937) Antagonist activity at CMV-fused ERbeta-LBD-GAL4-DBD (unknown origin) expressed in HEK293T cells assessed as inhibition of estradiol-induced receptor transactivation after 16 hrs by bright-Glo luciferase reporter gene assay
- ChEMBL_2370015 Uncompetitive inhibition of recombinant human full-length C-terminal His-tagged HSD17B13 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- ChEMBL_2370016 Uncompetitive inhibition of recombinant human full-length C-terminal His-tagged HSD17B11 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- ChEMBL_2370018 Uncompetitive inhibition of recombinant mouse full-length C-terminal His-tagged HSD17B13 using estradiol and NAD as substrate preincubated for 10 mins followed by substrate addition and measured after 40 mins by MALDI-TOF-MS analysis
- Estrogen Receptor Binding Assay and Cell-Based Transcriptional Assay The binding affinities were determined by a competitive radiometric binding assay using [3H]estradiol as tracer. The Kd for 3H-estradiol was determined by saturation binding to ER receptors. The IC50 values for compounds were converted to Ki using Cheng-Prusoff equation. The functional activity and selectivity was measured using a transcription assay in the cotransfected human prostate cancer PC3/ER (alpha or beta)-ERE cell line, and EC50 values were determined by computer fit to a concentration-response curve.
- ChEMBL_1497344 (CHEMBL3579293) Antagonist activity at human estrogen receptor alpha expressed in CHOK1 cells assessed as inhibition of beta-estradiol-induced protein interaction with steroid receptor co-activator peptide after overnight incubation by beta-galactosidase reporter gene assay
- ChEMBL_2369991 Inhibition of recombinant human full-length C-terminal His-tagged HSD17B13 expressed in HEK293 cells using estradiol as substrate preincubated for 30 mins followed by substrate addition and measured after 3 hrs by rapidfire MS/MS analysis
- ChEMBL_2370017 Uncompetitive inhibition of recombinant human full-length C-terminal His-tagged HSD17B13 expressed in HEK293 cells using estradiol as substrate preincubated for 30 mins followed by substrate addition and measured after 3 hrs by rapidfire MS/MS analysis
- Binding Assay The competition assay was performed in a 96-well plate (polystyrene*) which binds <2.0% of the total input [3H]-17beta-estradiol and each data point was gathered in triplicate. 100 uG/100 uL of the receptor preparation was aliquoted per well. A saturating dose of 2.5 nM [3H]17beta-estradiol+competitor (or buffer) in a 50 uL volume was added in the preliminary competition when 100x and 500x competitor were evaluated, only 0.8 nM [3H]17beta-estradiol was used. The plate was incubated at room temperature for 2.5 h. At the end of this incubation period 150 uL of ice-cold dextran coated charcoal (5% activated charcoal coated with 0.05% 69K dextran) was added to each well and the plate was immediately centrifuged at 99 g for 5 minutes at 4 C. 200 uL of the supernatant solution was then removed for scintillation counting. Samples were counted to 2% or 10 minutes, whichever occurs first.
- ChEMBL_1721239 (CHEMBL4136239) Antagonist activity at recombinant human His6-tagged ERalpha LBD (298 to 554 residues) expressed in Escherichia coli assessed as inhibition of estradiol-induced fluorescein-labeled PGC1alpha coactivator peptide binding after overnight incubation by Lantascreen TR-FRET assay
- ChEMBL_2113959 (CHEMBL4822809) Antagonist activity at ERalpha (unknown origin) expressed in human HEK293T cells assessed as inhibition of 17beta-estradiol-induced transcriptional activities by measuring ERE driven reporter gene expression measured after 24 hrs by dual luciferase reporter gene assay
- ChEMBL_2113960 (CHEMBL4822810) Antagonist activity at ERbeta (unknown origin) expressed in human HEK293T cells assessed as inhibition of 17beta-estradiol-induced transcriptional activities by measuring ERE driven reporter gene expression measured after 24 hrs by dual luciferase reporter gene assay
- Sulfatase Assay The extent of in vitro inhibition of sulfatase activities was assessed using intact monolayers of JEG-3 cells. Sulfatase activity was measured using [6,7-3H] EIS over 3 hrs, determined by measuring the total amount of 3H-labeled estrone and estradiol formed.
- Sulfatase Inhibition Assay The extent of in vitro inhibition of sulfatase activities was assessed using intact monolayers of JEG-3 cells. Sulfatase activity was measured using [6,7-3H] E1S over 3 hrs, determined by measuring the total amount of 3H-labeled estrone and estradiol formed.
- ChEMBL_1721240 (CHEMBL4136240) Antagonist activity at recombinant human His6-tagged ERalpha LBD (298 to 554 residues) D538G mutant expressed in Escherichia coli assessed as inhibition of estradiol-induced fluorescein-labeled PGC1alpha coactivator peptide binding after overnight incubation by Lantascreen TR-FRET assay
- ChEMBL_1721241 (CHEMBL4136241) Antagonist activity at recombinant human His6-tagged ERalpha LBD (298 to 554 residues) Y537S mutant expressed in Escherichia coli assessed as inhibition of estradiol-induced fluorescein-labeled PGC1alpha coactivator peptide binding after overnight incubation by Lantascreen TR-FRET assay
- Binding Assay The ER binding affinity of the compounds was determined using an in vitro competitive radioligand binding assay was [2,4,6,7-3H(N)]-Estradiol ([3H]E2), a natural high affinity ER ligand, and bacterially expressed GST fusion ER-alpha or ER-beta ligand binding domain (LBD) protein.
- ER-alpha Radioligand Binding Assay and ERE-Luciferase Reporter Assay. Radioligand binding assay was performed by using 96-well microtiterplates containing ER, 17beta-estradiol, and the test compound to be tested and SPAbeads. After incubation at room temperature for 2-4 h, the reaction was terminated by centrifugation. The radioactivity was counted in a Packard Topcount scintillation counter. Nonspecific binding was defined as the remaining radioactivity in the presence of 10 uM nonradioactive 17beta-estradiol. Assays were performed in triplicate. HELNalpha, a human cervix adenocarcinoma cell line derived from HeLa cells stably transfected with the reporter gene and the expression plasmid ERalpha, were used to quantify the antiestrogenic and estrogenic effects of compounds on ERE
- ER-alpha Radioligand Binding Assay and ERE-Luciferase Reporter Assay. Radioligand binding assay was performed by using 96-well microtiterplates containing ER, 17beta-estradiol, and the test compound to be tested and SPAbeads. After incubation at room temperature for 2-4 h, the reaction was terminated by centrifugation. The radioactivity was counted in a Packard Topcount scintillation counter. Nonspecific binding was defined as the remaining radioactivity in the presence of 10 uM nonradioactive 17beta-estradiol. Assays were performed in triplicate. HELNalpha, a human cervix adenocarcinoma cell line derived from HeLa cells stably transfected with the reporter gene and the expression plasmid ERalpha, were used to quantify the antiestrogenic and estrogenic effects of compounds on ERE.
- ER-beta Radioligand Binding Assay and ERE-Luciferase Reporter Assay. Radioligand binding assay was performed by using 96-well microtiterplates containing ER, 17beta-estradiol, and the test compound to be tested and SPAbeads. After incubation at room temperature for 2-4 h, the reaction was terminated by centrifugation. The radioactivity was counted in a Packard Topcount scintillation counter. Nonspecific binding was defined as the remaining radioactivity in the presence of 10 uM nonradioactive 17beta-estradiol. Assays were performed in triplicate. HELNbeta, a human cervix adenocarcinoma cell line derived from HeLa cells stably transfected with the reporter gene and the expression plasmid ERbeta, were used to quantify the antiestrogenic and estrogenic effects of compounds on ERE.
- Human ERalpha Reporter Assa All reagents used in this assay was supplied in the Human ERα Reporter Assay by Indigo Biosciences # IB00401. In an effort to screen selective estrogen receptor degraders (SERDs), the Human ERα Reporter Assay, supplied by Indigo Biosciences, was utilized to quantify antagonist functional activity against the human estrogen receptor. Reporter cells were thawed at 37° C. and added to pre-warmed to 37° C. cell recovery medium (CRM). Stock concentration of 17β-estradiol was serially diluted in CRM. Diluted 17β-estradiol was added to CRM containing reporter cells resulting in a working concentration of 1.6 nM (2×). Cells plus 17β-estradiol were dispensed in a kit-supplied white walled 96-well plate. Concentrated stocks of test compounds were diluted to 2× working concentrations in cell screening medium (CSM). 2× concentrated compounds were added to the plated cells in a dose-dependent manner resulting in a final concentration range of 1E-11 to 1E-5 M and a final 17β-estradiol concentration of 8E-10 M. Assay plates were incubated for 24 hours in a humidified 37° C. incubator. Culture medium was removed from the assay plates by inversion. Detection substrate and buffer was warmed to room temperature, mixed thoroughly, and immediately added to the assay plates. Assay plates were incubated for 15 minutes at room temperature protected from light. Luminescence was measured in a Synergy HTX luminescence plate reader. Data is processed utilizing GraphPad Prism 7 by graphing the relative light units measured at each compound concentration.
- Human ERalpha Reporter Assay All reagents used in this assay was supplied in the Human ERα Reporter Assay by Indigo Biosciences #IB00401. In an effort to screen selective estrogen receptor degraders (SERDs), the Human ERα Reporter Assay, supplied by Indigo Biosciences, was utilized to quantify antagonist functional activity against the human estrogen receptor. Reporter cells were thawed at 37° C. and added to pre-warmed to 37° C. cell recovery medium (CRM). Stock concentration of 17β-estradiol was serially diluted in CRM. Diluted 17β-estradiol was added to CRM containing reporter cells resulting in a working concentration of 1.6 nM (2×). Cells plus 17β-estradiol were dispensed in a kit-supplied white walled 96-well plate. Concentrated stocks of test compounds were diluted to 2× working concentrations in cell screening medium (CSM). 2× concentrated compounds were added to the plated cells in a dose-dependent manner resulting in a final concentration range of 1E-11 to 1E-5 M and a final 17β-estradiol concentration of 8E-10 M. Assay plates were incubated for 24 hours in a humidified 37° C. incubator. Culture medium was removed from the assay plates by inversion. Detection substrate and buffer was warmed to room temperature, mixed thoroughly, and immediately added to the assay plates. Assay plates were incubated for 15 minutes at room temperature protected from light. Luminescence was measured in a Synergy HTX luminescence plate reader. Data is processed utilizing GraphPad Prism 7 by graphing the relative light units measured at each compound concentration.
- Biochemical Antagonist Activity on Wild Type (WT) and Mutants Antagonistic potency of compounds was evaluated using LanthaScreen TR-FRET ERα Coactivator Assay (ThermoFisher) with modifications. It is a competition assay, where binding of a test compound to a complex comprised of (i) His6-ERα298-554 protein representing ERα ligand-binding domain, (ii) Tb-labeled His6 antibody, (iii) a fluorescein-labeled PGC1a coactivator peptide (EAEEPSLLKKLLLAPANTQ), and (iv) estradiol, results in a decrease of the TR-FRET signal due to dissociation of the coactivator peptide. His6-ERα298-554 proteins were expressed as WT or D538G or Y537S mutants in E. coli and purified by affinity chromatography. The assay works in a homogeneous mix-and-read format. In a typical experiment, a 4 μL mixture of 0.5 nM His6-ERα298-554, 0.5 nM Tb-labeled His6 antibody, 250 nM PGC1a peptide, and 3 nM estradiol (or 10 nM estradiol) in 100 mM potassium phosphate, pH 7.4, 0.01% Tween-20, 0.02% NaN3, 5 mM DTT, was added to 40 nL test compound in DMSO and incubated overnight at room temperature. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation.
- Estrone Detection Assay for Evaluation of HSD17beta13 Activity The liquid chromatography/mass spectrometry (LC/MS) estrone detection assay monitors the conversion of estradiol to estrone by HSD17B13. This assay was undertaken in a 96wp format (Eppendorf deep well Plate 96/500) in an 80 µl reaction volume containing: 4 µM of Estradiol (E2; Cayman; #10006315), 6 mM NAD+ (Sigma; #N0623) and 30 nM HSD17B13 enzyme (in-house; E. coli expressed His-tagged, purified, soluble protein) in a reaction containing 1 M potassium phosphate buffer pH 7.4, with 0.5% vehicle (DMSO). Reactions were incubated for 2 hours at 26.5° C., and estradiol (E2) conversion to estrone (E1) was quantitated by LC-MS/MS based analyte detection for both E2 and E1 using LCMS grade reagents.Reactions were terminated by the addition of two volumes of acetonitrile (MeCN; LCMS grade; CAS# 75/05/8) containing deuterated (D4)-E1 used as internal standard (Clear Synth; #CS-T-54273; 500 ng/mL final concentration). Samples were applied to pre-prepared Bond Elut-C18 extraction cartridges (3 mL; Agilent; #12102028), washed and eluted in MeCN. Eluates were dried under nitrogen and re-suspended in 60% methanol (LCMS grade methanol; CAS# 67/56/1) before submission for analysis. Aqueous linearity for E2 and E1 were included for quantification.
- Estrogen Receptor Binding Assay. Arzoxifene and its analogues were assayed in the standard ER competitive radioligand binding assay, using full length human recombinant ER-alpha and ER-beta with [3H]-estradiol. IC50 values were calculated from binding of the sample expressed as a percentage relative to E2 (50 nM, 100%). Relative binding affinity (RBA; relative to E2) was calculated from IC50 (E2)/IC50 (sample). The samples were assayed in triplicate at least five concentrations.
- HSD17B13 Inhibition Assay HSD17B13 uses the oxidized form of nicotinamide adenine dinucleotide (NAD+) as a cofactor during metabolism of beta-estradiol (substrate), converting NAD+ to the reduced form (NADH) and beta-estradiol to its product (estrone). Estrone production is quantified by LCMS, and used as a measure of HSD17B13 enzyme activity.HEK-cells stably expressing wild type human HSD17B13 were plated at 10,000 cells/well in 50 μL growth media (DMEM containing 10% heat inactivated FBS, 400 μg/ml geneticin, 1× L-Glutamine, and 1× Non-essential amino acids, Invitrogen 11965-092, 16140-071, 10131-027, 25030-081, 11140-050), into poly-D-lysine-coated 384-well plates (Corning Biocoat, 354663), and incubated overnight (with lid) at 37° C. (95% O2: 5% CO2). Following overnight incubation, intermediate compound plates (Greiner, 781280) containing 160 nL of 375×FAC test compound which had been serial diluted 1 in 3.162 in 100% DMSO for an 11 point concentration response curve, with duplicate points at each concentration, were diluted 1 in 187.5 with 30 μL warmed assay media (DMEM, 1× L-Glutamine, and 1× Non-essential amino acids) to give 2×FAC compound (80 μM top concentration) in 0.53% DMSO. Growth media was then removed from the cell plates and replaced with 10 μL of 2×FAC test compound, and incubated for 1 hour (with lid) at 37° C. (95% O2: 5% CO2), before the addition of 25 μM FAC beta-estradiol in assay media/0.2% DMSO. The reaction was incubated for 2 hours (with lid) at 37° C. (95% O2: 5% CO2), after which 10 μL of reaction was transferred from assay plate to a new 384-well deep-well plate (Matrix 4325) and diluted 1 in 10 with 90 μL of stop reagent (50% methanol in water containing internal standard 17b-estradiol-2,3,4-13C3). Amount of product (estrone) was then quantified by LCMS. Data expressed as product area ratio (PAR) were then normalized to control wells using Activity Base (IDBS).
- Radioligand Binding of Compounds to AR, GR and ER ReceptorsGR (human) (agonist radioligand) IM-9 cells (cytosol)[3H]dexamethasone 1.5 nM 1.5 nM triamcinolone (10 μM) 6 h 4° C. Scintillation counting (Clark, A. F et al. (1996) Invest. Ophtalmol. Vis. Sci., 37: 805-813).ER (nonselective) (human) (agonist radioligand) MCF-7 cells (cytosol)[3H]estradiol 0.4 nM 0.2 nM 17(3-estradiol (6 μM) 20 h 4° C. Scintillation counting(Parker, G. J et al. (2000) J. Biomol. Screen., 5: 77-88).AR (human) (agonist radioligand) LNCaP cells (cytosol)[3H]methyltrienolone 1 nM 0.8 nM mibolerone (1 μM) 24 h 4° C. Scintillation counting.Zava, D. T et al. (1979) Endocrinology, 104: 1007-1012.The results are expressed as a percent of control specific binding measured specific binding*100 control specific binding and as a percent inhibition of control specific binding 100-(measured specific binding*100) control specific binding obtained in the presence of compound.
- Estrogen Receptor Coactivator Assay It is a competition assay, where binding of a test compound to a complex comprised of (i) His6-ERα298-554 protein representing ERα ligand-binding domain, (ii) Tb-labeled His6 antibody, (iii) a fluorescein-labeled PGC1α coactivator peptide (EAEEPSLLKKLLLAPANTQ), and (iv) estradiol, results in a decrease of the TR-FRET signal due to dissociation of the coactivator peptide. His6-ERα298-554 proteins were expressed as WT or D538G or Y537S mutants in E. coli and purified by affinity chromatography. The assay works in a homogeneous mix-and-read format. In a typical experiment, a 4 μL mixture of 0.5 nM His6-ERα298-554, 0.5 nM Tb-labeled His6 antibody, 250 nM PGC1a peptide, and 3 nM estradiol in 100 mM potassium phosphate, pH 7.4, 0.01% Tween-20, 0.02% NaN3, 5 mM DTT, was added to 40 nL test compound in DMSO and incubated overnight at room temperature. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation. The antagonist activity with respect to estrogen receptors in this test is given by the concentration which inhibits 50% of the estrogen receptor activity (or IC50) in nM.
- HSD10B1 Enzymatic Blocking Assay To screen compounds of the disclosure for their ability to block HSD17B10 function, an enzymatic blocking assay was performed. For the assay, HSD17B10 activity was measured by detecting NADH formation from oxidation of Estradiol using a NADH Glo luminescence assay (Promega, #Cat. No. G9062). Compounds of the disclosure were evaluated in a dose titration assay where the compounds were titrated at 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B10 (Seq. ID NO: 5, expressed in mammalian cells) was added at a final concentration of 31.25 ng/well in a buffer containing 0.2M Tris-HCl, pH 7.5, into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 30 minutes at room temperature. The assay reaction was then initiated by addition of 25 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 500 μM NAD co-factor, and the reaction mixture was incubated for 3 hours at room temperature. DMSO at a concentration of 0.5% was used as a negative control and a no substrate control was used a positive control in the assay. Detection reagent was then added as per manufacturer's instructions, and the plate was subsequently incubated in the dark for 1 hour at room temperature, NADH generated was detected in the luminescence mode on the Envision Perkin Elmer plate reader. The IC50 values were determined using Dotmatics analysis software.
- HSD17B1 Enzymatic Blocking Assay To screen compounds of the disclosure for their ability to block HSD17B1 function, an enzymatic blocking assay was performed. For the assay, HSD17B1 activity was measured by detecting NADH formation from oxidation of Estradiol using a NADH Glo luminescence assay (Promega, #Cat. No. G9062). Compounds of the disclosure were evaluated in a dose titration assay where the compounds were titrated at 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B1 (SEQ ID NO: 2, expressed in mammalian cells) was added at a final concentration of 25 ng/well in a buffer containing 0.2M Tris-HCl, pH 7.5, into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 30 minutes at room temperature. The assay reaction was then initiated by addition of 25 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 500 μM NAD co-factor, and the reaction mixture was incubated for 3 hours at room temperature. DMSO at a concentration of 0.5% was used as a negative control and a ‘no substrate’ control was used a positive control in the assay. Detection reagent was then added as per manufacturer's instructions, and the plate was subsequently incubated in the dark for 1 hour at room temperature, NADH generated was detected in the luminescence mode on the Envision Perkin Elmer plate reader. The IC50 values were determined using Dotmatics analysis software.
- Enzyme Inhibition on the Conversion of E2 to E1 by 17beta-HSD1 For steady-state kinetic study of hybrid inhibitors, a Fluorolog 3 instrument was used to monitor the fluorescent signal of NADPH formed during estradiol oxidation, through which the substrate conversion is monitored. The excitation wavelength was 340 nm, and the emission wavelength was 460 nm. A standard curve monitoring the increase of fluorescence (cps) caused by an increase of NADPH revealed that the formation of 1 uM NADPH corresponds to a 316,000 cps increase. The Km for E2 in the absence of EM-1745, and four apparent Km values in the presence of 0.5, 2, 4, and 7 nM EM-1745 were obtained. Ki was further determined by a plot of Km vs. the inhibitor concentration.
- Competition Binding Assay Run the competition binding assay in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mM DTT, using 0.025 μCi per well 3H-estradiol (118 Ci/mmol, 1 mCi/mL), 7.2 ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7 ng/well ERβ receptor. Add the test compound at 10 different concentrations ranging from 10,000 nM to 0.5 nM, and determine nonspecific binding in the presence of 1 μM of 1713 estradiol. Incubate the binding reaction (140 μL) for 4 hours at room temperature, and then add cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4° C. and then centrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120 μL) of the mixture to another 96-well, white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) to each well. Seal the plates and shake vigorously on an orbital shaker. After an incubation of 2.5 hours, read the plates in a Wallac Microbeta counter.
- HSD17B2 Enzymatic Blocking Assay To screen compounds of the disclosure for their ability to block HSD17B2 function, an enzymatic blocking assay was performed. For the assay, HSD17B2 activity was measured by detecting NADH formation from oxidation of Estradiol using a NADH Glo luminescence assay (Promega, #Cat. No. G9062). Compounds of the disclosure were evaluated in a dose titration assay where the compounds were titrated at 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B2 (SEQ ID NO: 3, expressed in mammalian cells) was added at a final concentration of 2 ng/μL in a buffer containing 50 mM Tris-HCl, pH 7.5, Pluronic F-127 0.05%, Tween20 0.01%, BSA 0.01% into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 15 min at 20° C. The assay reaction was then initiated by addition of 1.8 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 650 μM NAD co-factor, and the reaction mixture was incubated for 45 min at 20° C. DMSO at a concentration of 0.5% was used as a negative control and a ‘no substrate’ control was used a positive control in the assay. Detection reagent was then added as per manufacturer's instructions, and the plate was subsequently incubated in the dark for 1 hour at room temperature, NADH generated was detected in the luminescence mode on the Envision Perkin Elmer plate reader.
- HSD17B4 Enzymatic Blocking Assay To screen compounds of the disclosure for their ability to block HSD17B4 function, an enzymatic blocking assay was performed. For the assay, HSD17B4 activity was measured by detecting NADH formation from oxidation of Estradiol using a NADH Glo luminescence assay (Promega, #Cat. No. G9062). Compounds of the disclosure were evaluated in a dose titration assay where the compounds were titrated at 10-point dose curve using an Echo-555 liquid handling dispenser. HSD17B4 (SEQ ID NO: 4, expressed in mammalian cells) was added at a final concentration of 2.5 ng/μL in a buffer containing 50 mM Tris-HCl, pH 7.5, Pluronic F-127 0.05%, Tween20 0.01%, BSA 0.01%, into a low volume white 384-well assay plate. Compounds were incubated with the enzyme for 15 min at 20° C. The assay reaction was then initiated by addition of 23.46 μM Estradiol substrate (Sigma, catalog #E2758-250MG, CAS: 50-28-2) and 1500 μM NAD co-factor, and the reaction mixture was incubated for 45 min at 20° C. DMSO at a concentration of 0.5% was used as a negative control and a ‘no substrate’ control was used a positive control in the assay. Detection reagent was then added as per manufacturer's instructions, and the plate was subsequently incubated in the dark for 1 hour at room temperature, NADH generated was detected in the luminescence mode on the Envision Perkin Elmer plate reader.
- UGT1A1 Inhibition Assay KCI838 was incubated at seven increasing concentrations with human UGT1A1-expressed Supersomes (0.25 mg/mL), alamethicin (25 μg/mL) and UDPGA (5 mM) in the presence of the probe substrate estradiol (10 μM) for 30 min at 37 C. The UGT1A1 inhibitor, atazanavir, was screened alongside the test compounds as a positive control. The reactions were terminated by quenching with one volume of methanol containing an analytical internal standard. The samples will be centrifuged at 5000 rpm for 10 min at 4 C. The metabolites were monitored by LC-MS/MS and a decrease in the formation of the metabolite compared to the vehicle control was used to calculate an IC50 value (test compound concentration which produces 50% inhibition).
- Binding Assay ERRα/ERRβ/ERα : In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
- Competition-Based Ligand Binding Assay and Transactivation Assay. Ligand binding was determined using a scintillation proximity assay with streptavidin-coated SPA beads (Amersham) and biotinylated receptor. Receptor-bound [3H]estrone was determined by scintillation counting (Perkin-Elmer). The binding IC50 value (the concentration of compound required for 50% inhibition of [3H]estrone binding to ER was calculated using XL-fit one-site dose response. To determine the agonist activity of the compounds on receptors, the full length receptors were stably expressed under ERE promoter with luciferase as reporter in Hela cells. The transactivation EC50 (the concentration of compound required to achieve 50% of transactivation caused by 10 nM estradiol) was calculated using XL-fit one-site dose response.
- HSD17b13 NAD(P)H-Glo Biochemical Assay HSD17b13 enzyme was diluted in 1× assay buffer to the desired enzyme concentration based on the specific activity of the enzyme lot. 20 uL of diluted enzyme was added to each well along with 2.5 uL of 10× inhibitor solution. Assay plate was incubated at RT for 20 minutes, and then 2.5 uL of a 10× substrate/cofactor mix was added to each well for a final concentration of 50 uM estradiol and 1 mMNAD+. Assay plate was incubated at 37° C. for 3 hours. NAD(P)H-Glo Detection System reagents were prepared according to manufacturer's specifications, and 25 uL was added to each well. After incubating for 1 hour at RT, luminescence was measured.
- Luciferase Assay Estrogen receptor-negative CV-1 kidney cells are maintained in Dulbecco's modified Eagle's medium with 4.5 g/L glucose supplemented with 10% fetal bovine serum and 100 units/ml penicillin-streptomycin at 37° C. in a humidified 5% CO2 atmosphere. The cells are then plated in 6-well dishes at a density of 2×10^5 cells per well in phenol-red free Dulbecco's modified Eagle's medium containing 10% charcoal-dextran-stripped fetal bovine serum. CV-1 cells are transfected using LipofectAMINE reagent according to the manufacturer's protocol. Transfections containing 1.5 μg of reporter plasmid (containing ERE-tk-luciferase containing a single ERE cloned upstream of the thymidine kinase promoter and luciferase gene) and 0.5 μg of either ERα or ERβ expression vector (containing CMV-ERα or CMV-ERβ full length coding sequence respectively). The next day, cells receive no treatment (controls) or are treated with estradiol alone (1 nM) or estradiol plus a compound of the invention (at varying concentrations). After 16-24 hours, cells are harvested and assayed for luciferase activity. At the outset, cell monolayers are washed twice with ice-cold phosphate-buffered saline and incubated for 15 minutes in 250 μl of 1× cell culture lysis reagent (Promega, Madison, Wis.). Cell extracts are transferred to a fresh tube and assayed using the luciferase assay system (Promega). For each assay, 10 μl of extract is diluted with 90 μl of 1× cell culture lysis reagent. Luminescence is read using an AutoLumat LB953 luminometer.
- Binding Assay In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
- ERalpha (Wild Type), ERalpha (Y537S Mutant) and ERbeta Competition Binding Assay The purpose of the following ER competition binding assays is to determine the affinity of a test compound against ERα (wild type), ERα (Y537S mutant), and ERβ.Run the competition binding assay in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mM DTT, using 0.025 μCi per well 3H-estradiol (118 Ci/mmol, 1 mCi/mL), 7.2 ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7 ng/well ERβ receptor. Add the test compound at 10 different concentrations ranging from 10,000 nM to 0.5 nM, and determine nonspecific binding in the presence of 1 μM of 17-β estradiol. Incubate the binding reaction (140 μL) for 4 hours at room temperature, and then add cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4° C. and then centrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120 μL) of the mixture to another 96-well, white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) to each well. Seal the plates and shake vigorously on an orbital shaker. After an incubation of 2.5 hours, read the plates in a Wallac Microbeta counter. Calculate the IC50 using a 4-parameter logistic curve fit and calculate % inhibition at 10 μM. Convert the IC50 values for the compound to Ki using Cheng-Prusoff equation.
- Competitive Binding Assay Competitive binding assays were performed by incubating rhER alpha (α) and beta 1 (β1) receptors with 10 nM [3H]estradiol (the radio ligand) in the presence or absence of increasing concentrations, 0.25 to 250,000 nM, of the phenolic test compounds of Tables 1 to 3 (nM is nano molar). Each data point is the average of at least two assays. Stock solutions of the compounds of Tables 1 to 3 were prepared at 10×E-2 M in 100% ethanol, water or DMSO (dimethyl sulfoxide). Compounds were diluted 10 fold in binding buffer and then 1:4 in the final assay mix. The final concentration of ethanol or DMSO in the assay well was 5%. The highest concentration of the hydrolysis test compound was 2.5×E-4 M (250,000 nM). The residual monomers of Tables 1 to 3 were tested at seven concentrations over log increments. The lowest concentration was 2.5×E-10 M (0.25 nM).
- Fluorescence Polarization Assay Peptides and the ERα LBD were diluted in 1× PBS buffer [140 mM NaCl, 2.7 mM KCl, 10 mM K2HPO4, and 2 mM KH2PO4 (pH 7.4)]. Peptide concentrations were determined by the absorbance at 492 nm, using a fluorescein extinction coefficient of 83000. Fluorescence polarization assays were conducted in 96-well round-bottom black opaque plates (Costar) using 2-fold serial dilutions of the ERα LBD, to final protein concentrations from 10 to 0.0098 μM, in PBS containing 0.1 mM DTT, 20 μM 17β-estradiol, 0.04 mg/mL BSA, and 100 nM fluorescein-labeled peptide. Solutions were incubated in the dark for 30 min at room temperature before being read on a PerkinElmer Fusion plate reader using a 485 nm fluorescein excitation filter and a 535 nm emission filter with a polarizer.
- ERalpha (Wild Type), ERalpha (Y537ERalpha (Wild Type), ERalph (Y537S Mutant) and ERbeta Competition Binding Assay The purpose of the following ER competition binding assays is to determine the affinity of a test compound against ERα (wild type), ERα (Y537S mutant), and ERβ.Run the competition binding assay in a buffer containing 50 mM HEPES, pH 7.5, 1.5 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mg/mL ovalbumin, and 5 mM DTT, using 0.025 μCi per well 3H-estradiol (118 μCi/mmol, 1 mCi/mL), 7.2 ng/well ERα (wild type), or 7.2 ng/well ERα (Y537S mutant) or 7.7 ng/well ERβ receptor. Add the test compound at 10 different concentrations ranging from 10,000 nM to 0.5 nM, and determine nonspecific binding in the presence of 1 μM of 17-β estradiol. Incubate the binding reaction (140 μL) for 4 hours at room temperature, and then add cold dextran-charcoal buffer (70 μL) (containing per 50 mL of assay buffer, 0.75 g of charcoal and 0.25 g of dextran) to each reaction. Mix the plates for 8 minutes on an orbital shaker at 4° C. and then centrifuge at 3000 rpm at 4° C. for 10 minutes. Transfer an aliquot (120 μL) of the mixture to another 96-well, white flat bottom plate (Costar) and add Perkin Elmer Optiphase Supermix scintillation fluid (175 μL) to each well. Seal the plates and shake vigorously on an orbital shaker. After an incubation of 2.5 hours, read the plates in a Wallac Microbeta counter. Calculate the IC50 using a 4-parameter logistic curve fit and calculate % inhibition at 10 μM. Convert the IC50 values for the compound to Ki using Cheng-Prusoff equation. The results of this assay demonstrate Examples 1, 1A, and 1B (and others) bind to recombinant ERα wild type and ERα mutant (Y537S) as shown in Table 7 below and Example 1B was also determined to bind to ERβ with a Ki (nM) ERβ competition of 0.11±0.07, n=3.
- Competitive Radioligand-Binding Assay Estrogen receptor (ERβ) binding affinity of the NRBAs was also determined using an in vitro competitive radioligand-binding assay with [3H]-estradiol ([3H]-E2, PerkinElmer), a high affinity ligand for both ERα and ERβ. The equilibrium dissociation constant (Kd) of [3H]-E2 was determined by incubating increasing concentrations of [3H]-E2 (0.01 to 10 nM) with bacterially expressed ERα or ERβ ligand binding domain (LBD) at 4° C. for 18 hours (h). Non-specific binding was determined by adding 1000 nM E2 to the incubation mixture. It was determined that the minimum concentration of [3H]-E2 required to saturate ERα and ERβ binding sites in the incubation mixture was 1 nM, respectively. The binding affinity of the NRBAs was determined under identical conditions by incubating increasing concentrations (3×10−2 to 1,000 nM) of ligand with isolated ER LBD and 1 nM [3H]-E2. Following incubation, bound and free [3H]-E2 were separated by using vacuum filtration with the Harvester (PerkinElmer). Briefly, the incubation mixture was filtered through a high affinity protein binding filter, and washed several times to remove any unbound radioactivity. The filter plate was air dried and sealed on the bottom. Scintillation cocktail was added to each well and the top of the plate was sealed. Radioactivity was counted in a TopCount NXT Microplate Scintillation Counter.
- ERα TR-FRET Test 1x Tris-HCl (Sigma, PHG0002) protein buffer was prepared and mixed for later use. The compound to be detected was prepared into a stock solution with a concentration of 2 mM and then subjected to serial 3-fold gradient dilution, resulting in a total of 10 concentrations. The diluted compounds were respectively added to a reaction plate by means of Echo 550, with 100 nL per well, and at the same time 5 nL of estradiol (Sigma, 491187; final concentration 1.5 nM) was added to each well.Preparation of 1x protein mixed solution: firstly, 2x GST-ERalpha-LBD (Invitrogen, A15677)/MAb anti-GST-Eu (Cisbio, 61GSTKLA) mixed solution was prepared according to the following table.Final Working Concentration ofconcentration concentration stock solutionSubstance (nM) (nM) (nM)GST-ERalpha-LBD 2 4 20100MAb anti-GST-Eu 2.5 ng/well 50 nl/well 50 ug/ml2x biotin-SRC2/streptavidin-XL665 (Cisbio, 610SAXLA) mixed solution was prepared.Final Working Concentration ofconcentration concentration stock solutionSubstance (nM) (nM) (nM)Biotin-SRC2 75 150 1000000Streptavidin-XL665 50 ng/well 50 nl/well 1 mg/mlThe above 2x GST-NR-LBD/ MAb anti-GST-Eu solution and 2x biotin-SRC2/streptavidin-XL665 solution were uniformly mixed at a volume ratio of 1:1; and the 1x protein mixed solution was added to each well of a 384-well plate, with 20 uL being added per well, the 384-well plate was put into a centrifuge and centrifuged at room temperature at 1000 rpm for 10 seconds, and it was taken out, then left to stand at room temperature for 3 h, and then read by EnVision multifunctional microplate reader.The values at 665 and 615 (nm) were read out, and with the value at 615 as the correction value, the final value was expressed as the value at 665/the value at 615.
- ER Transcription Assay (MCF7 Cells) The ER transcription assay is a reporter assay that is based on the ability of ER to induce transcription from a luciferase reporter gene containing estrogen response elements (EREs) in the promoter/enhancer region. When the reporter gene is transfected in MCF7 cells (containing endogenous ER), transcription is reflected by the level of luciferase expression.MCF7 cells are maintained in DMEM/F12 (Gibco, catalog number 11330) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, catalog number 100-106). A day before transfection, cells are split into a T75 flask at a cell density of 300,000 cells/mL (10 mL total) and allowed to attach overnight in a humidified CO2 incubator at 37° C.Next day, prior to transfection, media is switched to DMEM/F12 (Gibco, catalog number 21041) supplemented with 10% charcoal-stripped serum (Gemini Bio-Products, catalog number 100-119). MCF7 cells are then bulk transfected, using Lipofectin (Invitrogen, catalog number 18292) with the following plasmids: 7x-TK-ERE-Luc3 (ER reporter gene) and pCMV-Renilla (normalization control). Briefly, for each T75 flask, 32.5 μL of Lipofectin is added to 617.5 μL of OptiMEM (Gibco #11058) and incubated for 30 min at 37 C. Approximately 20 ug DNA is mixed in OptiMEM (Invitrogen) to a total volume of 650 μL. Following incubation, the OptiMEM-DNA mixture is added to the OptiMEM-Lipofectin mix and incubated for 15 minutes at 37° C. The DNA-Lipofectin mixture is then added directly to the T75 flask and the flask is returned to the incubator.After overnight incubation, compound is added to individual wells of a 96-well plate in a 10 μL volume of media at 10× concentration along with 1713 estradiol whose final concentration is 0.1 nM. Normally, DMSO (used as a vehicle) is included to achieve a final concentration of 0.1% when added to the cells. Transfected cells are trypsinized, resuspended in DMEM/F12/10% charcoal-stripped serum and added to the 96-well plate at 25,000 cells/well in 90 μL of media. The plate is then returned to the incubator for 24 hours.After incubation with compounds for 24 hours, Firefly and Renilla luciferase activities are measured to determine ER transcriptional activity. Media is removed from 96-well plates by decanting and blotting on paper towels. Cells are lysed with 40 ul/well of 1× passive lysis buffer (25 mM Tris Phosphate, 2 mM CDTA, 10% Glycerol, 0.5% Triton X-100 and 2 mM DTT before use) and allowed incubate at room temperature for 10 minutes.Firefly luciferase activity is measured by adding 30 ul Firefly luciferase assay buffer (20 mM Tricine, 0.1 mM EDTA, 1.07 mM (MgCO3)4 Mg(OH)2*5H2O, 2.67 mM MgSO4, 33.3 mM DTT, 270 μM Coenzyme A, 470 μM luciferin, 530 μM ATP, reconstituted) per well, followed by measuring light units using a luminometer (BMG labtech FLUOstar OPTIMA). One second total read time after a one second delay.Renilla luciferase activity is measured by adding 50 ul Renilla luciferase assay buffer (1.1M NaCl, 2.2 mM Na2EDTA, 0.22 M KxPO4 (pH 5.1), 0.44 mg/mL BSA, 1.3 mM NaN3, 1.43 uM coelenterazine, final pH adjusted to 5.0), per well, followed by measuring light units using a luminometer. One second total read time after one second delay. If Firefly luciferase signal is high, Renilla assay must be done an hour after the Firefly assay due to incomplete squelching of Firefly signal.