CLONING, EXPRESSION AND CHARACTERIZATION OF RHESUS MACAQUE TYPE-1 ANDTYPE-2 5ALPHA-REDUCTASE - EVIDENCE FOR MECHANISM-BASED INHIBITION BY FINASTERIDE

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus 5aRs share d 94% and 96% identity with the human type 1 and 2 isozymes, respectiv ely. Despite a four amino acid insertion at the N-terminal region of r hesus 5aR1, the biochemical properties of rhesus and human homologs ar e very similar with respect to pH optimum, K-m values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expec ted, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays t ime dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M-1 s(-1) and 5.2 x 10(5) M-1 s(-1). Inhi bition of rhesus 5aR2 with H-3-finasteride resulted in H-3 bound to th e enzyme which is not released by dialysis. Heat denaturation of the [ rhesus 5aR2:inhibitor] complex releases dihydrofinasteride, a breakdow n product presumably related to the NADP(+)-adduct previously identifi ed with the human 5aRs (Bull et al., Mechanism-based inhibition of hum an steroid 5 alpha-reductase by finasteride: Enzyme catalyzed formatio n of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J . Amer. Chem. Sec., 1996, 118, 2359-2365). Taken together, these resul ts provide good evidence that the rhesus macaque is a suitable model t o evaluate the pharmacological properties of finasteride and other 5aR inhibitors. (C) 1998 Elsevier Science Ltd. All rights reserved.