CHEMBL3889248

Inhibitory Activity Assay: The inhibition of CYP17A1 by the test compounds was evaluated using a recombinant enzyme. Human CYP17A1 was expressed in E. coli (Ehmer, P. B. et al.; J. Steroid Biochem. Mol. Biol., 75, 57-63 (2000)). The microsomal fraction and 140 μL of phosphate buffer (50 mM Na phosphate, 1 mM MgCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, pH 7.4) were preincubated separately with a mixture of progesterone (24.95 μM) and 3H-progesterone (0.05 μM, 101.3 Ci/mmol), 50 μM of an NADPH regeneration system (in phosphate buffer with 10 mM NADP+, 100 mM glucose 6-phosphate and 2.5 U of glucose 6-phosphate dehydrogenase) and the appropriate test substances (in 5 μl of DMSO) at 37° C. for 5 minutes. The reaction was started by addition of the enzyme and, after 30 minutes of incubation at 37° C., stopped by addition of 50 μl of 1N hydrochloric acid.The steroids were extracted with ethyl acetate. After evaporation of the organic phase, the steroids were taken up in acetonitrile.