CLONING, EXPRESSION AND FUNCTIONAL-CHARACTERIZATION OF TYPE-1 AND TYPE-2 STEROID 5-ALPHA-REDUCTASES FROM CYNOMOLGUS MONKEY - COMPARISONS WITH HUMAN AND RAT ISOENZYMES

The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two know n human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the sui tability of this species at the level of the molecular targets, a Cyno molgus monkey prostate cDNA library was prepared and screened using hu man SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA s equences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with th eir human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corre sponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Sub sequently, both monkey cDNAs were individually expressed in a mammalia n cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and ra t enzymes, the monkey type 1 and type 2 SRs were most active at neutra l and low pH, respectively. The results of inhibition studies with ove r 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocit y and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest th at the monkey SR isoenzymes are structurally and functionally comparab le on a molecular level to their respective human counterparts, suppor ting the relevance and use of the Cynomolgus monkey as a pharmacologic al model for in vivo evaluation of SR inhibitors.