7A-Methyl-Testosterone BDBM50423527 CHEMBL359619 7Alpha-Methyl-Testosterone
4-Methyl-Testosterone BDBM50423534 CHEMBL265925
6Alpha-Methyl-Testosterone CHEMBL260640 BDBM50423520
9A-Fluoro-Testosterone BDBM50423524 CHEMBL259599
BDBM50423514 CHEMBL411316 16Alpha-Hydroxy-Testosterone
(1S,2R,10R,11S,14S,15S)-14-hydroxy-2,15-dimethyltetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadec-6-en-5-one Testosterone US9682960, Testosterone Testosterone, 1 BDBM8885 17beta-Hydroxyandrost-4-en-3-one
17-Hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren-3-one (testosterone)17-Hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren-3-one testosterone CHEMBL386630 17-Hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren-3-one(testosterone) BDBM50025452 14-hydroxy-2,15-dimethyltetracyclo[8.7.0.02,7.011,15]heptadec-6-en-5-one 17-Hydroxy-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-cyclopenta[a]phenanthren-3-one (Testosterone)
Propionic acid (8R,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl ester BDBM50215709 testosteron propionate Propionic acid 10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl ester(Testosterone Propionate) CHEMBL1170 Propionic acid 10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl ester TESTOSTERONE PROPIONATE
- Al-Aboudi, A; Odeh, H; Khalid, A; Naz, Q; Choudhary, MI Butyrylcholinesterase inhibitory activity of testosterone and some of its metabolites. J Enzyme Inhib Med Chem 24: 553-8 (2009)
- Asami, T; Nishizawa, N; Matsui, H; Nishibori, K; Ishibashi, Y; Horikoshi, Y; Nakayama, M; Matsumoto, S; Tarui, N; Yamaguchi, M; Matsumoto, H; Ohtaki, T; Kitada, C Design, synthesis, and biological evaluation of novel investigational nonapeptide KISS1R agonists with testosterone-suppressive activity. J Med Chem 56: 8298-307 (2013)
- Asami, T; Nishizawa, N; Matsui, H; Takatsu, Y; Suzuki, A; Kiba, A; Terada, M; Nishibori, K; Nakayama, M; Ban, J; Matsumoto, S; Tarui, N; Ikeda, Y; Yamaguchi, M; Kusaka, M; Ohtaki, T; Kitada, C Physicochemically and pharmacokinetically stable nonapeptide KISS1 receptor agonists with highly potent testosterone-suppressive activity. J Med Chem 57: 6105-15 (2014)
- Marhefka, CA; Moore, BM; Bishop, TC; Kirkovsky, L; Mukherjee, A; Dalton, JT; Miller, DD Homology modeling using multiple molecular dynamics simulations and docking studies of the human androgen receptor ligand binding domain bound to testosterone and nonsteroidal ligands. J Med Chem 44: 1729-40 (2001)
- ChEMBL_814102 Reversible inhibition of CYP3A4-mediated conversion of testosterone to 6-beta-hydroxy-testosterone after 30 mins
- ChEBML_210239 Binding affinity towards testosterone receptor
- ChEMBL_51920 Inhibition of Cytochrome P450 3A4 with testosterone
- ChEMBL_581617 Inhibition of CYP3A4 using testosterone as substrate
- ChEMBL_620426 Inhibition of CYP3A4 using testosterone as a substrate
- ChEMBL_673953 Inhibition of CYP3A4 using testosterone as substrate
- ChEMBL_701747 Inhibition of CYP3A4 uisng testosterone substrate
- ChEMBL_792912 Displacement of [3H]testosterone from AR
- ChEMBL_827974 Inhibition of CYP3A4 using testosterone as substrate
- ChEMBL_858103 Inhibition of CYP3A4 using testosterone as substrate
- ChEMBL_1980041 Inhibition of human CYP3A4 using testosterone as substrate
- ChEMBL_643970 Inhibition of human CYP3A4-mediated testosterone oxidation
- ChEMBL_857030 Reversible inhibition of CYP3A4 using testosterone as substrate
- ChEMBL_1772941 Inhibition of human supersome CYP3A4 using testosterone as substrate assessed as testosterone 6beta-hydroxylation after 30 mins by HPLC-UV detection
- ChEMBL_1432845 Inhibition of CYP3A4 (unknown origin) using testosterone substrate
- ChEMBL_1435707 Inhibition of CYP3A4 (unknown origin) using testosterone substrate
- ChEMBL_1442889 Inhibition of CYP3A4 (unknown origin) using testosterone substrate
- ChEMBL_1513925 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1613542 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1696494 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1780160 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1794906 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1855510 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1865168 Inhibition of human recombinant CYP3A4 using testosterone as substrate
- ChEMBL_1866304 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1898402 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1983162 Inhibition of recombinant human CYP3A4 using testosterone as substrate
- ChEMBL_1983163 Inhibition of recombinant human CYP3A5 using testosterone as substrate
- ChEMBL_1983174 Inhibition of recombinant human CYP3A4 using testosterone as substrate
- ChEMBL_1983175 Inhibition of recombinant human CYP3A5 using testosterone as substrate
- ChEMBL_1983186 Inhibition of recombinant human CYP3A4 using testosterone as substrate
- ChEMBL_1983187 Inhibition of recombinant human CYP3A5 using testosterone as substrate
- ChEMBL_2020183 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_2024275 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_2115923 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_2148408 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_2156398 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_50540 Inhibition of human placental cytochrome P450 19A1 with testosterone
- ChEMBL_51034 Inhibition of human placental cytochrome P450 19A1 with testosterone
- ChEMBL_51036 Inhibition of human placental cytochrome P450 19A1 with testosterone
- ChEMBL_741042 Inhibition of human CYP3A4 in human liver microsomes assessed as formation of 6beta-hydroxy-testosterone using testosterone as substrate by high throughput mass spectrometry in absence of NADPH
- ChEMBL_1468060 Inhibition of CYP3A4 (unknown origin) using testosterone as marker substrate
- ChEMBL_1589887 Inhibition of human placental microsomal aromatase using testosterone as substrate
- ChEMBL_1667019 Inhibition of human liver microsomes CYP3A4 using testosterone as substrate
- ChEMBL_1748557 Inhibition of CYP3A4-mediated testosterone metabolism in human liver microsomes
- ChEMBL_1817350 Reversible inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_1896396 Inhibition of human aromatase using [1beta,2beta3H]-]testosterone as substrate
- ChEMBL_1914413 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_1935816 Time-dependent inhibition of human CYP3A4 using testosterone as substrate
- ChEMBL_2056542 Inhibition of CYP3A4 in human liver microsome using testosterone as substrate
- ChEMBL_2125914 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_2151594 Inhibition of CYP3A4 in human liver microsome using testosterone as substrate
- ChEMBL_2165881 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_813719 Inhibition of CYP3A4 in human liver microsomes co-incubated with testosterone
- ChEMBL_875802 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_1283600 Inhibition of CYP3A4 in human liver microsomes assessed as 6beta-hydroxylation of testosterone
- ChEMBL_1462161 Reversible inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_1554473 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone 6beta-hydroxylation
- ChEMBL_1747252 Inhibition of human CYP3A4 using testosterone as substrate preincubated for 30 mins
- ChEMBL_1817351 Time dependent inhibition of CYP3A4 (unknown origin) using testosterone as substrate
- ChEMBL_2100795 Direct inhibition of CYP3A4 in human liver microsomes using testosterone as substrate
- ChEMBL_2100796 Direct inhibition of CYP3A5 in human liver microsomes using testosterone as substrate
- ChEMBL_210247 Binding constant of Testosterone-5 alpha-reductase activity at pH 7.4
- ChEMBL_320884 Binding affinity to androgen receptor by incubation with GST-hARLBD and [3H]testosterone
- ChEMBL_487546 Displacement of [3H]testosterone from wild type human androgen receptor
- ChEMBL_753596 Reversible inhibition of CYP3A4 assessed as conversion of testosterone to 6-beta-hydroxytestosterone
- ChEMBL_859942 Inhibition of human CYP3A4 using testosterone as substrate after 45 mins
- ChEMBL_741043 Inhibition of human CYP3A4 in human liver microsomes assessed as formation of 6beta-hydroxy-testosterone using testosterone as substrate by high throughput mass spectrometry preincubated for 30 mins in presence of NADPH
- ChEBML_1683160 Inhibition of CYP3A4 in human liver microsomes using testosterone, midozolam or nifedipine as substrates
- ChEMBL_1625607 Inhibition of human Cyp3A4 using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_1713962 Inhibition of human CYP3A4 using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_210244 Inhibition of Testosterone-5 alpha-reductase activity at pH 7.4 from rat
- ChEMBL_2125132 Inhibition of human CYP3A4 using testosterone as a substrate by LC/MS/MS analysis
- ChEMBL_2157118 Inhibition of human CYP3A4 using testosterone as substrate by fluorescence plate reader analysis
- ChEMBL_225417 In vitro inhibitory activity against rat testosterone 5 alpha reductase was determined
- ChEMBL_738395 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone hydroxylation
- ChEMBL_738408 Mechanism based inhibition of human cytochrome P450 3A5 measured by testosterone hydroxylation
- ChEMBL_753598 Time dependent inhibition of CYP3A4 assessed as conversion of testosterone to 6-beta-hydroxytestosterone
- ChEMBL_813040 Inhibition of human CYP3A4 using testosterone as substrate by LC/MS/MS analysis
- ChEMBL_1362982 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone conversion to 6-hydroxy testosterone preincubated for 10 mins followed by NADPH addition measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1447575 Inhibition of CYP3A4 in human hepatocytes using testosterone as substrate by HPLC/MS/MS method
- ChEMBL_1539997 Inhibition of rat liver type 1 5alpha-reductase assessed as transformation of testosterone to dihydrotestosterone
- ChEMBL_1539998 Inhibition of human prostate type 2 5alpha-reductase assessed as transformation of testosterone to dihydrotestosterone
- ChEMBL_1584554 Inhibition of human aromatase using testosterone as substrate incubated for 1 hr by HTRF assay
- ChEMBL_1618865 Inhibition of CYP3A4 in pooled human hepatic microsomes using testosterone substrate in presence of NADPH
- ChEMBL_210241 Inhibition of rat testosterone-5 alpha-reductase activity at pH 6.6 from rat
- ChEMBL_2165351 Inhibition of recombinant human CYP3A4 using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_50691 Inhibition of Cytochrome P450 19A1 against testosterone at 1.5 uM (Km=0.13 uM)
- ChEMBL_51017 Inhibition of Cytochrome P450 19A1 against testosterone at 1.5 uM (Km=0.13 uM)
- ChEMBL_572594 Inhibition of CYP3A4 in human liver microsomes assessed as inhibition of testosterone 6-beta-hydroxylation
- ChEMBL_726892 Displacement of [3H]testosterone from Sprague-Dawley rat AR by liquid scintillation counting
- ChEMBL_738394 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone beta-hydroxylation
- ChEMBL_821319 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by fluorescence detection assay
- ChEMBL_942984 Inhibition of CYP3A4/5 in human liver microsomes assessed as reduction in testosterone-beta-hydroxylation
- ChEMBL_1491569 Inhibition of human recombinant CYP3A5 using testosterone as substrate assessed as testosterone 6-beta-hydroxylation preincubated up to 30 mins with NADPH followed by substrate addition measured after 10 mins by LC/MS/MS analysis
- ChEBML_50700 Inhibition of cytochrome P450 19A1 of human placental microsomes with [1-beta,2beta-3H]testosterone
- ChEMBL_1347924 Inhibition of human hepatocyte microsome CYP3A4 using testosterone as substrate by HPLC/MS/MS method
- ChEMBL_1441437 Inhibition of rat liver 5alpha-reductase type 1 assessed as conversion of [3H]testosterone to dihydrotestosterone
- ChEMBL_1441438 Inhibition of human prostate 5alpha-reductase type 2 assessed as conversion of [3H]testosterone to dihydrotestosterone
- ChEMBL_1485703 Inhibition of CYP3A4 in human liver microsomes using testosterone substrate by LC-MS/MS method
- ChEMBL_1492827 Inhibition of CYP3A4 activity in human liver microsomes using testosterone as a substrate by LC/MS analysis
- ChEMBL_1520473 Inhibition of human placental aromatase in presence of [1beta,2beta-3H] testosterone by Thompson and Siiteri method
- ChEMBL_1653831 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by HPLC-MS/MS method
- ChEMBL_1705360 Inhibition of CYP3A4 in human hepatocyte microsomes using testosterone substrate by HPLC/MS/MS method
- ChEMBL_1705370 Inhibition of CYP19 in human hepatocyte microsomes using testosterone substrate by HPLC/MS/MS method
- ChEMBL_1979603 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_2051671 Inhibition of CYP3A4 in human liver microsomes using testosterone substrate by LC-MS/MS analysis
- ChEMBL_2062192 Inhibition of human liver microsomes CYP3A4 using testosterone as substrate by MUX-MS/MS analysis
- ChEMBL_2110160 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_2149012 Inhibition of human CYP3A4 using testosterone as substrate in presence of NADPH incubated for 15 to 45 mins
- ChEMBL_2157126 Time dependent inhibition of human CYP3A4 using testosterone as substrate by fluorescence plate reader analysis
- ChEMBL_322421 Androgen antagonistic activity as inhibitory concentration against testosterone-induced SC-3 cell proliferation
- ChEMBL_327080 Displacement of [3H]-[1-beta,2beta]-testosterone from 5alpha reductase2 in human prostrate homogenate
- ChEMBL_50552 Inhibition of human Placental Cytochrome P450 19A1 in vitro using human placental microsomes and testosterone
- ChEMBL_738373 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738375 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738376 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738377 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738379 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738380 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738381 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738383 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738384 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738385 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738388 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_738389 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation
- ChEMBL_817424 Inhibition of CYP3A4 using testosterone as substrate in human liver microsome for 20 mins by HPLC analysis
- ChEMBL_821298 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_854487 Inhibition of human CYP3A4 in liver microsomes assessed as testosterone 6beta-hydroxylation after 48 hrs
- ChEMBL_873340 Time dependent inhibition of CYP3A4-mediated testosterone-6-beta hydroxylation in human liver microsome
- ChEMBL_940630 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by LC-MS-MS analysis
- ChEBML_36132 Dissociation constant against GST-hARLBD was measured in SC-3 cell by using [3H]testosterone as radioligand
- ChEMBL_1655304 Inhibition of CYP3A4 using in human liver microsomes using testosterone as substrate after 5 to 15 mins
- ChEMBL_1667020 Inhibition of human liver microsomes CYP3A4 using testosterone as substrate preincubated with compound followed by substrate addition
- ChEMBL_2100805 Time-dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins
- ChEMBL_2100806 Time-dependent inhibition of CYP3A5 in human liver microsomes using testosterone as substrate preincubated for 30 mins
- ChEMBL_36057 In vitro inhibitory concentration against Aromatase was determined using [1-beta,2beta-3H]testosterone as radioligand
- ChEMBL_36135 Binding affinity against GST-hARLBD was measured in SC-3 cell by using [3H]testosterone as radioligand
- ChEMBL_738411 Mechanism based inhibition of rabbit cytochrome P450 3A6 measured by testosterone and progesterone 6-beta hydroxylation
- ChEMBL_753597 Time dependent inhibition of CYP3A4 assessed as conversion of testosterone to 6-beta-hydroxytestosterone preincubated for 30 mins
- ChEMBL_850907 Inhibition of human CYP3A4 using testosterone as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_1458157 Inhibition of human CYP3A4 using testosterone as substrate after 10 mins by SPE-MS analysis in presence of NADPH
- ChEMBL_1487665 Reversible inhibition of testosterone 6beta-hydroxylase activity of human recombinant CYP3A4 in presence of human P450 oxidoreductase and b5
- ChEMBL_1487667 Reversible inhibition of testosterone 6beta-hydroxylase activity of human recombinant CYP3A5 in presence of human P450 oxidoreductase and b5
- ChEMBL_1497736 Inhibition of human recombinant CYP3A4 using testosterone as substrate after 1 hr by LC-MS/MS analysis
- ChEMBL_1512777 Inhibition of CYP3A4 in human liver microsomes assessed as reduction in testosterone 6beta-hydroxylation by LC-MS/MS method
- ChEMBL_1584556 Inhibition of human placental microsomal aromatase using testosterone as substrate assessed as formation of estradiol after 10 mins
- ChEMBL_1584799 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 5 mins by LC-MS analysis
- ChEMBL_1624780 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1627005 Antagonist activity at androgen receptor in human MDA-MB-453 cells assessed as inhibition of testosterone-induced transactivation
- ChEMBL_1653051 Inhibition of human prostate 5-alpha reductase 2 assessed as decrease in conversion of testosterone to dihydrotestosterone by chromatographic method
- ChEMBL_1659464 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1712694 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1912369 Inhibition of CYP3A4 in human liver microsomes at 1 uM using testosterone as substrate by LC-MS/MS analysis
- ChEMBL_1923418 Inhibition of human CYP3A4 expressed in supersomes assessed as testosterone 6beta-hydroxylation after 15 mins by HPLC analysis
- ChEMBL_609086 Inhibition of CYP3A4-mediated hydroxylation of testosterone in human liver microsomes in NADPH co-factor system by RP-HPLC
- ChEMBL_699332 Inhibition of human prostate 5alpha-reductase type 2 assessed as formation dihydrotestosterone from [4-14C] testosterone
- ChEMBL_790052 Inhibition of recombinant human CYP3A4 assessed as conversion of testosterone into 6-hydroxytestosterone after 30 mins by HPLC analysis
- ChEMBL_796573 Inhibition of human 17beta-HSD3 expressed in HeLa cells assessed as conversion of 4-androstene-3,17-dione to testosterone
- ChEMBL_977682 Displacement of [3H]-Testosterone from human AR expressed in 293 cells after 16 hrs by scintillation counting
- ChEMBL_1366898 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_1445200 Inhibition of human CYP3A4 testosterone site in human liver microsomes using midazolam as substrate by LC-MS/MS analysis
- ChEMBL_1496402 Inhibition of CYP3A4 in human liver microsomes using testosterone substrate incubated for 30 mins by LC-MS/MS method
- ChEMBL_1578569 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 10 mins by LC/MS/MS analysis
- ChEMBL_1616181 Inhibition of CYP3A4 in human liver microsomes using testosterone/midazolam as substrate in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1624792 Reversible inhibition of CYP3A4 in human liver microsomes using testosterone as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2035475 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 30 mins by LC-MS/MS analysis
- ChEMBL_2046450 Time-dependent inhibition of CYP3A4 in human liver microsomes using testosterone in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2066474 Inhibition of human liver microsome CYP3A4 using testosterone as substrate incubated for 20 mins by LC-MS/MS analysis
- ChEMBL_2109895 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 10 mins by LC/MS/MS analysis
- ChEMBL_2157144 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate by TDI- high-throughput based mass spectrometry method
- ChEMBL_2158993 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 30 mins by LC-MS/MS analysis
- ChEMBL_738390 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation and erythromycin N-demethylation
- ChEMBL_738393 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation using a recombinant system
- ChEMBL_790073 Inhibition of human B-lymphoblastoid cell microsomal CYP3A4 assessed as testosterone 6beta-hydroxylation preincubated for 5 mins with substrate
- ChEBML_204741 Inhibitory activity against human 5-alpha-reductase type 2 using 18213 3H testosterone 210 nM as substrate
- ChEMBL_1438887 Displacement of [3H]testosterone from androgen receptor (unknown origin) expressed in 293 cells after 16 hrs by scintillation counting
- ChEMBL_1487045 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 5 to 30 mins by LC-MS/MS analysis
- ChEMBL_1578010 Inhibition of CYP3A4 in human liver microsomes using testosterone/midazolam as substrate after 30 mins by LC/MS/MS analysis
- ChEMBL_1619558 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 10 mins in presence of NADP by rapidfire/MS analysis
- ChEMBL_1785086 Inhibition of CYP3A4 in human liver microsomes using testosterone/midazolam as substrate after 30 mins by LC-MS/MS method
- ChEMBL_1803976 Inhibition of CYP3A4 in human liver microsomes using midazolam/testosterone as substrate after 10 to 30 mins by LC/MS analysis
- ChEMBL_1973582 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2073306 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2101777 Inhibition of CYP3A4 in human liver microsomes using midazolam or testosterone as substrate substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2150741 Inhibition of CYP3A4 in human pooled liver microsomes using midazolam and testosterone as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_50701 In vitro inhibition of cytochrome P450 19A1 from human placental microsomes with 2.5 uM [1-beta,2beta-3H]testosterone
- ChEMBL_738392 Mechanism based inhibition of human cytochrome P450 3A4 measured by testosterone 6-beta hydroxylation using human liver microsomes
- ChEMBL_975807 Inhibition of human 17beta-HSD5 expressed in HEK293 cells using androstenedione as substrate assessed as testosterone synthesis after 4 hrs
- ChEBML_204888 Inhibitory activity against rat Steroid 5-alpha-reductase type I using 18213 3H testosterone 210 nM as substrate
- ChEBML_50537 In vitro inhibitory concentration against human placental Cytochrome P450 19A was determined using [1-beta,2beta-3H]testosterone as radioligand
- ChEMBL_1713508 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone 6beta-hydroxylation after 4 to 40 mins in presence of NADPH by LCMS analysis
- ChEMBL_1869499 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 10 mins in presence of NADP by LC-MS/MS analysis
- ChEMBL_1992483 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 10 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_2022078 Time-dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate and in presence of co-factor by LC-MS/MS analysis
- ChEMBL_204742 Inhibitory activity against human Steroid 5-alpha-reductase type 2 using 18213 3H testosterone 210 nM as substrate
- ChEMBL_2075863 Inhibition of CYP3A4 in human pooled liver microsomes using testosterone as substrate measured up to 20 mins by UHPLC-MS/MS analysis
- ChEMBL_50537 In vitro inhibitory concentration against human placental Cytochrome P450 19A was determined using [1-beta,2beta-3H]testosterone as radioligand
- ChEMBL_775988 Displacement of [3H]testosterone from androgen receptor in Sprague-Dawley rat prostate gland after 2 hrs by liquid scintillation counting
- ChEMBL_975806 Inhibition of human 17beta-HSD5 expressed in human CWR22R cells using androstenedione as substrate assessed as testosterone synthesis after 4 hrs
- Scintillation Proximity Assay (SPA) SPA beads conjugated to anti-mouse IgG were incubated overnight in buffer with anti-testosterone monoclonal antibody. The enzymatic reaction was monitored by its conversion of 4-androstene-3, 17-dione to testosterone. The amount of [14C] testosterone produced in each well was determined by scintillation counting on a Perkin Elmer TopCount instrument and collected as DPMs. The data was then analyzed and percent inhibition and/or IC50 values, based on 10 point serially diluted curves in duplicate or triplicate, were calculated using Microsoft Excel and XLFit programs.
- ChEBML_306524 Inhibition of [1-beta-2beta-3H]- -testosterone binding to human steroid 5-alpha-reductase type 2 of BPH tissue at 10 uM
- ChEMBL_1492470 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone 6beta-hydroxylation after 3 mins by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1492480 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone 6beta-hydroxylation preincubated for 15 mins by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1520476 Inhibition of human placental aromatase assessed as conversion of 3H2O from [1beta,2beta-3H] testosterone after 20 mins by scintillation spectrometer analysis
- ChEMBL_1658362 Inhibition of microsomal CYP3A4 (unknown origin) using testosterone as substrate preincubated for 5 mins followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_1661010 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 10 mins followed by NADPH addition measured after 10 mins
- ChEMBL_1667937 Inhibition of CYP3A4 in human liver microsomes using midazolam/testosterone as substrate after 30 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1797911 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate after 30 mins in presence of NADPH measured by LC-MS/MS analysis
- ChEMBL_1859913 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate measured after 10 mins in presence of NADPH by UHPLC-MS/MS analysis
- ChEMBL_2022781 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate measured after 5 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2046448 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate measured after 15 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_204910 Evaluated for the inhibitory activity against human steroid 5-alpha-reductase type 2 from human BPH tissue at 210 nM of testosterone
- ChEMBL_2100086 Inhibition of CYP3A4 in human liver microsomes assessed as reduction in testosterone 6beta-hydroxylation activity by high-pressure liquid chromatography-tandem mass spectrometry
- ChEMBL_2101739 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_675520 Antiandrogen activity against androgen receptor in androgen-dependent mouse SC3 cells assessed as inhibition of testosterone-induced cell proliferation by WST1 assay
- ChEMBL_900593 Inhibition of Homo sapiens (human) aromatase assessed as 3H2O formation using [1beta,2beta-3H]testosterone as substrate by liquid scintillation counting analysis
- ChEMBL_942992 Antagonist activity at androgen receptor in androgen-dependent mouse SC3 cells assessed as inhibition of testosterone-induced cell growth after 3 days
- ChEMBL_1449353 Inhibition of Sprague-Dawley rat liver steroid 5-alpha-reductase assessed as inhibition of testosterone conversion to dihydrotestosterone incubated for 30 mins by HPLC method
- ChEMBL_1574682 Inhibition of CYP3A4 in human liver microsomes using testosterone and midazolam as substrate preincubated for 30 mins followed substrate addition by LC-MS/MS analysis
- ChEMBL_1673854 Inhibition of CYP3A4 in human liver microsomes assessed as decrease in formation of 6beta-hydroxytestosterone from testosterone after 10 mins by LC-MS/MS analysis
- ChEMBL_1701329 Inhibition of CYP3A4 in human liver microsomes assessed as decrease in formation of 6beta-hydroxytestosterone from testosterone after 10 mins by LC-MS/MS analysis
- ChEMBL_2118301 Reversible inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 10 mins followed by NADPH addition by LC/MS/MS analysis
- ChEMBL_775863 Inhibition of CYP3A4 in human liver microsomes assessed as inhibition of oxidative metabolism of testosterone after 45 mins by MS analysis in the presence of 1 mmol NADPH
- ChEMBL_1491683 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by substrate addition by LC-MS/MS analysis
- ChEMBL_1491685 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 90 mins followed by substrate addition by LC-MS/MS analysis
- ChEMBL_1492496 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate assessed as residual enzyme activity after 2 to 10 mins by LC-MS/MS analysis
- ChEMBL_1575837 Inhibition of 17betaHSD3 (unknown origin) transfected in human LNCAP cells assessed as conversion of [14C]-4-androstene-3,17-dione into [14C]-testosterone after 3 hrs
- ChEMBL_1614025 Inhibition of CYP3A4 in human liver microsomes assessed as 6-beta-hydroxytestosterone metabolite formation using testosterone as substrate incubated for 30 mins by HPLC-MS method
- ChEMBL_1677535 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate upto 10 uM after 5 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1896395 Inhibition of human placental microsome aromatase using [1beta,2beta3H]-]testosterone as substrate measured up to 21 mins in presence of NADPH by liquid scintillation spectrometer method
- ChEMBL_1972623 Inhibition of CYP3A4/5 in human liver microsomes using testosterone as substrate measured for 10 to 30 mins in presence of NADPH regenerating system by UPLC-MS analysis
- ChEMBL_2124316 Inhibition of CYP3A4 in human liver microsomes assessed as testosterone 6-beta-hydroxylation reaction incubated for 30 mins in presence of NADP by LC-MS/MS analysis
- ChEMBL_1462490 Inhibition of CYP3A4 in pooled mixed-gender human liver microsomes using testosterone substrate in presence of NADPH pre-incubated for 3 mins by HPLC-UV detection method
- ChEMBL_159371 Binding affinity against Progesterone receptor in human TE85 osteosarcoma cells was determined using (Z)-[125I]-17-alpha-(2-iodovinyl)-19-nor-testosterone as radioligand
- ChEMBL_1764609 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate pretreated for 5 mins followed by NADPH addition and measured after 5 mins by LC-MS analysis
- ChEMBL_1817314 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 10 mins followed by NADPH addition and measured for 15 mins by LC-MS/MS analysis
- ChEMBL_2016125 Inhibition of CYP3A4 (unknown origin) using testosterone as substrate preincubated for 5 mins followed by addition of NADPH measured after 20 mins by LC-MS/MS analysis
- ChEMBL_225213 Inhibitory concentration against human recombinant steroid 5-alpha-reductase type 2 in stably transfected chinese hamster ovary 1827 cells using [3H]testosterone
- ChEMBL_487550 Antagonist activity at wild type human recombinant androgen receptor assessed as inhibition of testosterone-induced growth of mouse androgen dependent SC3 cells by WST-1 method
- ChEMBL_565719 Inhibition of human 17beta-HSD5 expressed in HEK293 cells assessed as enzyme-mediated transformation of [14C]-4-androstene-3,17-dione in to [14C]-testosterone after 18 hrs
- ChEMBL_823391 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 3 mins prior to NAPDH-addition measured after 30 mins by RP-HPLC analysis
- ChEMBL_1491687 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by substrate addition by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1491689 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 90 mins followed by substrate addition by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1688700 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 15 mins measured after 8 mins in presence or absence of NADPH by LC/MS/MS analysis
- ChEMBL_1849385 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH addition measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1902240 Inhibition of CYP3A in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH addition and measured for 10 to 30 mins by LC-MS/MS analysis
- ChEMBL_1902876 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2022083 Inhibition of human CYP3A4 in human liver microsomes using testosterone as substrate and in presence of co-factor preincubated for 20 mins followed by substrate addition by LC-MS/MS analysis
- ChEMBL_205065 Inhibitory concentration against human recombinant steroid 5-alpha-reductase type I in stably transfected chinese hamster ovary (CHO) 1827 cells using [3H]testosterone.
- ChEMBL_2091855 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH addition and measured after 5 mins by LC-MS/MS analysis
- ChEMBL_2093986 Inhibition of human CYP3A4 in human pooled liver microsomes in presence of testosterone as substrate preincubated for 0 mins in presence of NADPH followed by addition of substrate by LC-MS/MS analysis
- ChEMBL_2093988 Inhibition of human CYP3A4 in human pooled liver microsomes in presence of testosterone as substrate preincubated for 30 mins in presence of NADPH followed by addition of substrate by LC-MS/MS analysis
- ChEMBL_2109894 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate incubated for 5 mins followed by NADPH addition and measured after 5 mins by LC/MS/MS analysis
- ChEMBL_2124740 Inhibition of CYP3A4 in pooled human liver microsomes using midazolam or testosterone as substrate preincubated for 30 mins in presence of NADPH followed by substrate addition by LC-MS/MS analysis
- ChEMBL_762463 Inhibition of 17Beta-HSD3 expressed in intact HEK293 cells assessed as transformation of [14C]-4-androstene-3,17-dione into [14C]-testosterone in presence of NADPH after 1 hr incubation
- ChEMBL_1274566 Inhibition of 17beta-HSD3 in rat testes microsomal fraction using [14C]-4-androstene-3,17-dione as substrate assessed as testosterone formation after 2 hrs by thin layer chromatography
- ChEMBL_1488503 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by 10-fold dilution and substrate addition by LC-MS/MS analysis
- ChEMBL_1488505 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 90 mins followed by 10-fold dilution and substrate addition by LC-MS/MS analysis
- ChEMBL_1512779 Time dependent inhibition of CYP3A4 in human liver microsomes assessed as reduction in testosterone 6beta-hydroxylation incubated for 60 mins in presence of NADPH generating system by LC-MS/MS method
- ChEMBL_1797910 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by substrate/NADPH addition measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1870803 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH cofactor addition and measured after 10 mins by LC-MS/MS analysis
- ChEMBL_2025116 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 5 mins followed by NADPH generating system addition and measured after 120 mins by LC-MS/MS
- ChEMBL_851741 Inhibition of recombinant CYP3A4 NF-14 expressed in Escherichia coli assessed as formation of 6-beta-hydroxytestosterone using testosterone as substrate after 5 mins by HPLC based UV method
- ChEMBL_1486112 Inhibition of testosterone 6beta-hydroxylase activity of human recombinant CYP3A4 in presence of human P450 oxidoreductase and b5 assessed as decrease in enzyme activity preincubated for 30 mins by LC-MS/MS analysis
- ChEMBL_1486114 Inhibition of testosterone 6beta-hydroxylase activity of human recombinant CYP3A5 in presence of human P450 oxidoreductase and b5 assessed as decrease in enzyme activity preincubated for 30 mins by LC-MS/MS analysis
- ChEMBL_1660998 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 20 mins followed by substrate addition measured after 30 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1904874 Inhibition of 17beta-HSD3 (unknown origin) expressed in human LNCAP cells assessed as reduction in [14C]-testosterone formation from [14C]-4-androstene-3,17-dione measured after 1 hr
- ChEMBL_1904878 Inhibition of 17beta-HSD3 in Sprague-Dawley rat testes microsomal fraction assessed as reduction in [14C]-testosterone formation from [14C]-4-androstene-3,17-dione measured after 2 hrs
- ChEMBL_1488507 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by 10-fold dilution and substrate addition by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1488509 Time dependent inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 90 mins followed by 10-fold dilution and substrate addition by LC-MS/MS analysis in presence of NADPH
- ChEMBL_1491679 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate assessed as unbound inhibitor concentration required for half maximal enzyme inactivation after 2 to 10 mins by LC-MS/MS analysis
- ChEMBL_2080684 Inhibition of CYP3A4 in human liver microsomes using testosterone as substrate preincubated for 10 mins in presence of NADPH generating system followed by substrate addition and measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1657628 Inhibition of 17-beta-HSD3 (unknown origin) expressed in human LNCaP cells using [14C]-4-androstene-3,17-dione as substrate assessed as reduction of [14C]-testosterone level after 1 hr by TLC method
- ChEMBL_1660935 Inhibition of CYP3A4 in human liver microsomes assessed as enzyme-mediated metabolite formation using testosterone as substrate incubated for 5 mins followed by NADPH addition measured after 30 mins by LC-MS/MS analysis
- ChEMBL_1972583 Inhibition of CYP3A4/5 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by substrate addition and measured after 10 to 40 mins in absence of NADPH regeneration system by UPLC-MS analysis
- ChEMBL_1972624 Inhibition of CYP3A4/5 in human liver microsomes using testosterone as substrate preincubated for 30 mins followed by substrate addition and measured after 10 to 40 mins in presence of NADPH regeneration system by UPLC-MS analysis
- Inhibition Study on the Interconversion of Testosterone to Androstenedione (4-dione) A radioactive assay was used for the enzyme kinetics in the presence of EM1404 at different concentrations for its Ki determination. Reactions were initiated by the addition of enzyme to the reaction mixture containing substrate and test compounds. After the reaction, the steroids were extracted, separated by TLC migration, and analyzed by phosphorimaging. The Km for testosterone oxidation in the absence of EM-1404, and four apparent Km values in the presence of 1, 3, 10, and 25 nM EM-1404, were determined. Ki value was further determined by a plot of Km vs. the inhibitor concentration.
- Aromatase Assay The enzyme activity was assayed by measuring the 3H-labeled H2O formed from [1beta,2beta-3H]testosterone during aromatization. After incubation, the reaction was terminated, and the charcoal-adsorbed steroids were separated by centrifugation. Aliquots of the supernatant were assayed for the presence of 3H-labeled H2O by counting using a liquid scintillation spectrometer.
- CYP19 Assay The enzyme activity was assayed by measuring the 3H-labeled H2O formed from [1beta,2beta-3H]testosterone during aromatization. After incubation, the reaction was terminated, and the charcoal-adsorbed steroids were separated by centrifugation. Aliquots of the supernatant were assayed for the presence of 3H-labeled H2O by counting using a liquid scintillation spectrometer.
- CYP19 Assay The enzyme activity was assayed by measuring the 3H-labeled H2O formed from [1beta, 2beta-3H] testosterone or [1beta-3H] androstenedione during aromatization. After incubation, the reaction was terminated, and the charcoal-adsorbed steroids were separated by centrifugation. Aliquots of the supernatant were assayed for the presence of 3H-labeled H2O by counting using a liquid scintillation spectrometer.
- Inhibition Assay (Testosterone Hydroxylase) To gain insight into the selectivity of the synthetic compounds, nicotine, nicotine related alkaloids and nicotine metabolites for inhibition of other CYPs, we examined the major CYP present in human liver (i.e., CYP 3A4). That the CYP2A6 inhibitors showed low or no inhibitory activity against CYP3A4 suggests that the inhibitors examined selectively inhibited CYP2A6.
- In Vitro Inhibition Assay This work was performed at the MDS Pharma Services, Pharmacology Laboratories, Taiwan. The assay was an in vitro evaluation of the ability of an extract or a pure compound to inhibit the steroid 5alpha -reductase enzyme from metabolizing testosterone into dihydrotestosterone. This is an enzyme-immunoassay (EIA) for quantitative determination of testosterone in human serum or plasma. The significance of this type of inhibition is that it can lead to eradication of benign prostatic hyperplasia (BPH). Two distinct isozymes are found in mice, rats, monkeys and humans: type 1 and II. Each of these isozymes is differentially expressed in tissues and developmental stages. In human, type 1 steroid 5alpha -reductase is predominant in the sebaceous glands of most regions of skin, including scalp and liver and is responsible for approximately one third of circulating DHT. Inhibitors of steroid 5alpha -reductase may be of benefit in the treatment of androgenetic alopecia.
- Inhibition Assay Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration=0.275%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate testosterone (50 μM) for 5 min at 37° C. The selective CYP3A4 inhibitor, ketoconazole, is screened alongside the test compounds as a positive control.
- Inhibition Assay The potencies of the compounds as inhibitors of human hepatic cytochromes P450 of the CYP3A subfamily (particularly CYP3A4) were assessed using well-characterized selective activities: midazolam 1'-hydroxylase (Kronbach, T., et al. Mol. Pharmacol. 36, 89-96 [1989]) and testosterone 6beta -hydroxylase (Waxman, D. J., et al. Arch. Biochem. Biophys. 263, 424-436, [1988]). For midazolam hydroxylase determination the final concentrations of microsomal protein and terfenadine substrate were 0.25 mg/mL and 2.5 uM, respectively, and the LC-MS internal standard was 1alpha -hydroxytriazolam. For testosterone hydroxylase activity the final microsomal protein concentration was 0.5 mg/mL, the substrate concentration was 50 uM and the LC-MS internal standard was D7-labeled 6beta -hydroxytestosterone. After incubation at 37 C. for 5 minutes, metabolic reactions were terminated by treatment with quench solution containing the appropriate internal standard.
- CYP3A4 Inhibition Assay The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
- Receptor Binding Assay Using cytosol from progesterone receptor-expressing insect cells (Hi5), competitive binding to the progesterone receptor was determined from the ability to displace 3H-progesterone as reference substance from the receptor. If a compound has an affinity corresponding to progesterone, this corresponds to a competition factor (CF) of 1. CF values greater than 1 are characterized by a lower affinity for the progesterone receptor, and CF values of less than 1 are characterized by higher affinity. The test was carried out as test above, with the following modifications: cytosol from androgen receptor-expressing insect cells (Hi5) was used, and the reference substance was 3H-testosterone.
- CYP Inhibition Assay Incubation of phenacetin, diclofenac, dextromethorphan, mephenotoin, albendazole and testosterone with human liver microsomes in the presence of in each case eight different concentrations of a compound formula (B) (as potential inhibitor) is carried out on a incubator shaker at 37 C. Standard incubation mixtures comprise NADPH and substrates in 100 mM phosphate buffer (pH 7.4) in a total volume of 200 μl. Test compound are dissolved in acetonitrile. Incubated with pooled human liver microsomes at 37° C. for a defined time. The reactions are stopped by adding 100 μl of acetonitrile in which a suitable internal standard is always present. Precipitated proteins are removed by centrifugation, and the supernatants analyzed by LC-MS/MS.
- Cyp Inhibition For Cyp inhibition, human liver microsomes from BD Gentest were incubated with Compound 028 or Compound 032 (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01 uM) and substrate (CYP1A2: Phenacetin at 30 uM; CYP2C9: Diclofenac at 10 uM; CYP2C19: S-Mephenytoin at 35 uM; CYP3A4: Midazolam at 5 uM and Testosterone at 80 uM; CYP2D6: Bufuralol at 10 uM) for the following incubation times: CYP1A2, 2C9, 2D6: 10 minutes, 37° C.; CYP2C19: 45 minutes, 37° C.; CYP3A4: 5 minutes, 37° C. Substrate conversion was measured by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Inhibition was calculated by curve fitting in Graph Pad Prism.
- Cyp inhibition For Cyp inhibition, human liver microsomes from BD Gentest were incubated with Compound 028 or Compound 032 (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01 μM) and substrate (CYP1A2: Phenacetin at 30 μM; CYP2C9: Diclofenac at 10 μM; CYP2C19: S-Mephenytoin at 35 μM; CYP3A4: Midazolam at 5 μM and Testosterone at 80 μM; CYP2D6: Bufuralol at 10 μM) for the following incubation times: CYP1A2, 2C9, 2D6: 10 minutes, 37° C.; CYP2C19: 45 minutes, 37° C.; CYP3A4: 5 minutes, 37° C. Substrate conversion was measured by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Inhibition was calculated by curve fitting in Graph Pad Prism.
- CYP450 Inhibition Assay The ability of the R and S enantiomers of (4-fluorophenyl)(4-((5-methyl-1H-pyrazol-3-yl)amino)quinazolin-2-yl)methanol to inhibit the common drug metabolizing isoforms of cytochrome P450 (CYP) was evaluated against the following isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. The compounds were incubated in duplicate with eight test compound concentrations (final DMSO concentration of 0.20%) with human liver microsomes (0.25 or 0.50 mg/mL) and NADPH (1 mM) in the presence of CYP isoform specific probe substrates (phenacetin, bupropion, taxol, diclofenac, mephenyloin, dextromethorphan, testosterone) at the Km for 10-20 minutes at 37° C. Selective CYP isoform inhibitors (furafulline, ticlopidine, quercetin, sulfaphenazole, ticlopidine, quinidine, ketoconazole) were screened alongside the test compounds as positive controls.
- Inhibition Assay The CYP3A4 inhibition assays were conducted using Human Liver Microsomes purchased from Invitrogen and designed to screen potential inhibitors of Cytochrome P450 in physiological condition. Initially the following reagents/mixtures were prepared: (i) Assay buffer: 0.1 M Phosphate buffer pH—7.4 (ii) Cofactor: 15 mM stock was prepared in assay buffer. Final concentration in assay—1.5 mM (iii) Substrate—50 mM DMSO stock was prepared for testosterone. From this a 10 mM sub-stock was prepared in MeCN. Further, a working stock solution of 700 μM was prepared in assay buffer. Final concentration in assay—70 μM (iv) Enzyme: 20 mg/mL stock was provided by manufacturer. Final concentration in assay was 0.5 mg/mL. At the start of the experiment, various concentrations of compound (7 different concs.) or positive control (Ketoconazole at a single concentration) were prepared in assay buffer. For 100 μL of final reaction system, 2.5 μL of HLM (20 mg/ml), 50 μL of test compound/reference compound from each concentration was added. Subsequently, 10 μL of substrate (testosterone 700 μM) and 10 μL of Cofactor (NADPH; 15 mM) were added. The volume was increased to 100 μL by adding assay buffer. DMSO concentration was kept as 0.5% uniform across all the reactions. The reaction was then allowed to incubate for 45 min at 37° C. After completion of the incubation period, the reaction was terminated by addition of 200 μL of chilled MeCN containing internal standard (Dexamethasone). The samples were than centrifuged and supernatants were analyzed using LCMS/MS.
- CYP Activity Assay Inhibition of hepatic cytochromes P450 was assessed in human liver microsomes using a substrate-specific approach of monitoring metabolites formed by each specific CYP enzyme. Metabolic reactions included phenacetin-O-deethylation (1A2), tolbutamide methylhydroxylation (2C9), S-mephenytoin 4'-hydroxylation (2C19), dextrome-thorphan-O-deethylation (2D6), and testosterone-6-β-hydroxylation (3A4). Each substrate was added at a concentration less than or equal to the Km for themetabolic pathway, and microsomal protein concentrations and assay incubation times were optimized for each reaction to ensure linear metabolite formation based on initial rates (Table 1). Microsomes were suspended in phosphate buffer and incubated at 37 °C in the presence of probe substrates. Reactions were initiated by the addition of an NADPH-regenerating buffer system and were quenched at the appropriate times using acetonitrile. Samples were centrifuged and concentrations of metabolites assessed by LC/MS.
- Androgen Receptor Binding Assay Androgen receptor (AR) binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats by competition binding assay (Schilling K. and Liao S., The Prostate, 1984; 5(6):581-588). Cytosol preparations and 1 nM [3H]mibolerone were incubated with increasing concentrations of test compounds. To determine non-specific binding, parallel incubations were carried out using excess of unlabelled testosterone. After incubation, bound and free steroids were separated by treatment with dextran-coated charcoal suspension. Bound radioactivity was determined by counting of supernatant fraction in scintillation fluid. Radioactivity was measured using a Microbeta counter. All data points were done as quadrublicates. Dissociation constant of [3H]mibolerone for rat androgen receptor was determined by saturation binding assay obtained from ventral prostates of castrated rats essentially as described (Isomaa V. et al., Endocrinology, 1982; 111(3):833-843).
- Binding Assay Androgen receptor (AR) binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats by competition binding assay (Schilling K. and Liao S., The Prostate, 1984; 5(6):581-588). Cytosol preparations and 1 nM [3H]mibolerone were incubated with increasing concentrations of test compounds. To determine non-specific binding, parallel incubations were carried out using excess of unlabelled testosterone. After incubation, bound and free steroids were separated by treatment with dextran-coated charcoal suspension. Bound radioactivity was determined by counting of supernatant fraction in scintillation fluid. Radioactivity was measured using a Microbeta counter. All data points were done as quadrublicates. Dissociation constant of [3H]mibolerone for rat androgen receptor was determined by saturation binding assay obtained from ventral prostates of castrated rats essentially as described (Isomaa V. et al., Endocrinology, 1982; 111(3):833-843).
- Discontinuous Radiometric Assay Compounds may be evaluated as selective reversible inhibitors of AKR1C3 by screening them against homogeneous recombinant AKR1C1-AKR1C4 expressed in E. coli. In each case, a discontinuous radiometric assay may be used to monitor the inhibition of progesterone reduction (20-ketosteroid reduction) catalyzed by AKR1C1, the inhibition of Δ4-AD reduction (17-ketosteroid reduction) catalyzed by AKR1C3, and the inhibition of 5α-DHT reduction (3-ketosteroid reduction) catalyzed by AKR1C2 and AKR1C4 (by measuring the formation of 20α-hydroxyprogesterone, testosterone or 3α-androstanediol by radiochromatography). Secondary screens of the compounds of interest include: (a) a full-screen against all nine human recombinant AKR enzymes to ensure there are no-intended off-target effects (in this context AKR1B10 (retinal reductase; SEQ ID NO:5) has been shown to be potently inhibited by N-phenylanthranilates) (Endo et al., 2010, Biol. Pharm. Bull. 33:886-90); (b) a screen against COX-1 and COX-2 to reaffirm that compounds do not act as NSAIDs; and (c) an expanded screen against other nuclear receptors (especially other steroid hormone receptors).
- AR Antagonism Antagonism of test compounds for AR was measured by reporter gene assay in human embryonic kidney (HEK293) cells stably transfected with an expression vector encoding full-length human AR and androgen responsive luciferase reporter gene construct (hAR/HEK293 cells). To determine antagonism for hAR, the cells were treated simultaneously with increasing concentrations of the test compound and submaximal concentration of testosterone (usually 0.45 nM). The final DMSO concentration was 1%. All test compounds were studied in triplicates. The cells were incubated for 24 before measurement of luciferase activity using Luciferase Assay System (Promega Corporation).Agonism of test compounds in AR overexpressing cells was measured by reporter gene assay in HEK293 cells stably transfected with an expression vector encoding full-length human AR and androgen responsive luciferase reporter gene construct. A clone expressing high levels of androgen receptor (5 times more than AR levels in AR-HEK293 cells) was selected to study agonism in AR overexpressing cells. To determine agonism, the cells were treated with increasing concentrations of the test compound. The final DMSO concentration was 1%. The test compounds were studied in triplicates and luciferase activity was determined as described above.
- Enzyme Activity Assay Test Example 4: 100 mM PBS buffer was formulated, which was then used to formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The 5× concentration of the compound working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). The 5× concentration of ketoconazole working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). Dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.20 μl of 2.5 mg/ml microsome solution, 20 μl of 50 μM testosterone working solution, 20 μl of MgCl2 solution and 20 μl of the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, different reaction systems for each concentration) were taken respectively and mixed well. For the positive control group, the compound was replaced with the same concentration of ketoconazole. The mixture together with 5 mM NADPH solution were pre-incubated at 37° C. for 5 minutes. After 5 minutes, 20 μl of NADPH were added to each well, the reaction was started and incubated for 30 minutes. All the incubated samples were present in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard were added to all samples, mixed well, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant were taken and analyzed by LC-MS/MS.
- Enzyme Activity Assay Test Example 5: 100 mM PBS buffer was formulated, which was then used to formulate 2.5 mg/ml microsome solution and 5 mM NADPH solution. The 5× concentration of the compound working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). The 5× concentration of quinidine working solution was diluted with PBS gradient (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). Dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.20 μl of 2.5 mg/ml microsome solution, 20 μl of 50 μM testosterone working solution, 20 μl of MgCl2 solution and 20 μl of the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, different reaction systems for each concentration) were taken respectively and mixed well. For the positive control group, the compound was replaced with the same concentration of quinidine. The mixture together with 5 mM NADPH solution were pre-incubated at 37° C. for 5 minutes. After 5 minutes, 20 μl of NADPH were added to each well, the reaction was started and incubated for 30 minutes. All the incubated samples were present in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard were added to all samples, mixed well, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant were taken and analyzed by LC-MS/MS.
- CYP MUX (3A4) RI Compound dilutions and assay-ready plates were prepared on a TTP Labtech mosquito HTS. Assay conduction was fully automated on a customized Screening Platform from Caliper (now PerkinElmer) containing a Mitsubishi robotic plate handler, Liconic incubators, a Caliper Zephyr® liquid handling workstation equipped with temperature-controlled deck positions, a Biotek MultiFlo dispenser and an Agilent PlateLoc heat sealer. Assay plates were Corning Costar 384 well PP plates. High throughput mass spectrometric readout was performed on a RapidFire® 300 system coupled to an AB Sciex API 4000™ triple quadrupole device. CYP isoform 3A4 was incubated in a separate reaction of 50 μL final volume. 25 μL of HLM (human liver microsomes, BD UltraPool™ 150, 0.25 mg/mL final concentration) and the respective substrate, testosterone (75 μM) for 3A4, in potassium phosphate buffer (100 mM, pH=7.4) were added to 250 nL of stamped compound solution (10 mM in DMSO). The reactions were started upon addition of 25 μL of a co-factor solution containing magnesium chloride (3.3 mM), glucose-6-phosphate (3.3 mM), glucose-6-phosphate dehydrogenase (1.4 units) and NADP (1 mM) in potassium phosphate buffer (100 mM, pH=7.4) and incubated on deck at 37° C. for 10 min. 8 μL of each reaction were transferred to the same readout plated filled with 48 μL of stop solution containing internal standards (concentration in final readout plate), 6-hydroxytestosterone-D7 (0.5 μM), 4′-hydroxydiclofenac-D4 (0.2 and dextrorphan-D3 (0.01 in acetonitrile with 0.5% formic acid. After heat sealing, plates were stored at −20° C. for at least 30 min, centrifuged and subjected directly to RapidFire/MS analysis.