Stereochemistry of the functional group determines the mechanism of aromatase inhibition by 6-bromoandrostenedione.

A selective inhibitor of aromatase (estrogen synthetase) would be a useful pharmacological tool with potential therapeutic application. We have found that 6 alpha-bromoandrostenedione (6 alpha-BrA) is a competitive inhibitor of human placental aromatase with respect to androstenedione, with an apparent Ki of 3.4 nM, while 6 beta-BrA is a mechanism-based irreversible inhibitor with an apparent Ki of 0.8 microM and a kinact of 0.025 min-1. Aromatase activity was measured by tritium release into water from the 1 beta position of [1(-3)H,4(-14)C]androstenedione in reaction mixtures containing NADPH and the aromatase. Time-dependent inhibition was assessed by preincubation of inhibitors with either the 900 X g placental pellet or placental microsomes in the presence of NADPH. Aliquots were taken at intervals, diluted, and assayed for aromatase activity with androstenedione and additional NADPH. The time-dependent inhibition by 6 beta-BrA was dependent on the concentration of this compound and the presence of NADPH, while the addition of excess substrate in the preincubation mixture hindered the inactivation. Both epimers were ineffective in inhibiting rabbit liver microsomal drug-metabolizing activities in a competitive or time-dependent manner. This indicates a high selectivity of 6-BrA inhibition among P-450 cytochromes. These and other 6-substituted androgens may be useful probes into the nature of the active site and mechanism of action of aromatase.