Development of a simple and rapid assay for the evaluation of inhibitors of human 17alpha-hydroxylase-C(17,20)-lyase (P450cl7) by coexpression of P450cl7 with NADPH-cytochrome-P450-reductase in Escherichia coli.

P450c17 is a microsomal enzyme catalyzing the last step in androgen biosynthesis. As inhibitors of P450c17 are promising drug candidates for the treatment of prostate cancer, it was our goal to develop a new cellular assay for the in vitro evaluation of potential inhibitors. Human P450c17 was expressed in E. coli and hydroxylase activity was determined using 1,2[3H]-progesterone. As the activity was low (1.7 pmol/min/mg protein), due to a lack of the requisite electron transfer partner NADPH-cytochrome-P450-reductase (NADPH-P450-reductase), coexpression of both the enzymes had to be performed. For that purpose, a plasmid was constructed which encoded human P450c17 and rat NADPH-P450-reductase in a transcriptional unit. This strategy led to a 100-fold increase in P450cl7 activity (175 pmol/min/mg protein). Time, pH and temperature dependence of progesterone conversion of this new monooxygenase system was determined. The K(M) of progesterone was 2.75 microM. An assay procedure for the evaluation of inhibitors was established and modified for high throughput screening using 96-well plates. Selected compounds were tested for their inhibitory activity using this whole cell assay. The data was compared to the results obtained in microsomal testicular preparations.