Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17 alpha-hydroxylase-C-17,C-20-lyase inhibitors

We have designed and synthesized a number of cytochrome P450 17 alpha-hydro xylase-C-17,C-20-lyase (P450c17) inhibitors with the aim of inhibiting andr ogen synthesis. To select the most potent inhibitors, we initially used hum an testicular microsomes, which have a high level of expression of this enz yme. However, due to lack of availability of human tissue and variability a mong the samples, we utilized recombinant human enzyme expressed in Escheri chia coli. We designed a simple and economical protocol based on the report that recombinant bovine P450c17 can be functionally active in live bacteri a In the assay we report here, we substituted high-performance liquid chrom atography product isolation with a rapid biochemical acetic acid releasing assay and utilized intact P450c17-expressing E. coli for the source of the enzyme. Enzymatic parameters of the bacterial system (K-m = 5.1 x 10(-7) M, V-max = 15.0 pmol/min/mg) were similar to those of human testicular micros omes (K-m = 4.8 x 10(-7) M, V-max = 40.0 pmol/min/mg), and our compounds di splayed a similar pattern of inhibition in both systems. This new system is a fast, reliable, and reproducible method for screening P450c17 inhibitors . Furthermore, it eliminates our dependence on human tissue and potential d ata fluctuations caused by variations in enzymatic activity between donors. (C) 1999 Academic Press.