17-ALPHA-(N-TERT-BUTYLCARBAMOYL)-4-AZA-5-ALPHA-ANDROSTAN-1-EN-3-ONE IS AN ACTIVE-SITE-DIRECTED SLOW TIME-DEPENDENT INHIBITOR OF HUMAN STEROID 5-ALPHA-REDUCTASE-1

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one (finasteride), which has been approved for treatment of benign prostatic hyperplasia, is shown here to be a slow time-dependent inhibitor of human steroid 5 alpha-reductase isozyme 1. This inhibition is characterized by an initial, fast step where the inhibitor binds to the enzyme followed by a slow step that leads to a final enzyme-inhibitor complex (EI*). No recovery of activity from this EI* complex was observed after dialysis for 3 days. The formation of EI* is diminished in the presence of a competitive, reversible inhibitor, indicating that the inhibition is active site-directed. At 37 degrees C and pH 7.0, the rate constant for the second, slow inhibition step, k(3) is (1.40 +/- 0.04) x 10(-3) s(-1) and the pseudo-bimolecular rate constant, k(3)/K-i, is (4.0 +/- 0.3) X 10(3) M(-1) s(-1), This latter rate constant is less than the value of 2.7 x 10(5) M(-1) s(-1) determined for the inhibition of Sa-reductase 2 by finasteride [Faller, B., Farley, D., and Nick, H. (1993) Biochemistry 32, 5705-5710]. The 1,2-double bond within the A ring of finasteride may be the key structural element required for the slow step, since two other 4-azasteroids, 5 alpha-23-methyl-4-aza-21-norchol-1-en-3,20-dione and 17 beta-(N,N-diethylcarbamoyl)-4-aza-5 alpha-androstan- 1-en-3-one, which share the same A ring structure with finasteride, are also slow inhibitors of 5 alpha-reductase 1, whereas 17 beta-(N,N-diethylcarbamoyl)-4-methyl-4-aza-5 alpha and 17 beta-(N,N-diethylcarbamoyl)-4-aza-5 alpha-androstan-3-one, which lack the 1,2-double bond in the A ring of finasteride, do not show slow inhibition kinetics. These data suggest covalent modification of 5 alpha-reductase by finasteride.