Development of a simple and rapid assay for the evaluation of inhibitors of human 17α-hydroxylase-C17,21-lyase (P450c17) by coexpression of P450c17 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, iii was our goal to develop; new cellular assay for the in vitro evaluation of potential inhibitors. Human P450c17 was expressed in E. toll and hydroxylase activity was determined using 1.2[H-1]-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 P450c17 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 muM. 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. (C) 2001 Elsevier Science Ltd. All rights reserved.