Active site-directed inhibition of rabbit cytochrome P-450 1 by amino-substituted steroids.

Abstract

A variety of amino-substituted steroids were investigated as inhibitors of the rabbit hepatic, steroid 21-hydroxylase, cytochrome P-450 1. We reasoned that a steroid analog of pregnenolone capable of mimicking the binding of C21-steroids to the enzyme at the active site and bearing an amine moiety on the 17 beta-side chain would be a potent inhibitor if the amine were free to interact with the heme iron. Our studies reveal that 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC) is a tightly-bound inhibitor of cytochrome P-450 1-catalyzed reactions (Ki less than 1 nM). Spectral differences elicited by 22-ABC indicate that when bound to the enzyme, the amine moiety of 22-ABC is coordinated to the heme iron. In contrast, several other hepatic cytochrome P-450s which mediate distinct regio-specific routes of metabolism for progesterone or pregnenolone remain largely unaffected at concentrations of 22-ABC that exceed by 2 orders of magnitude that required to inhibit cytochrome P-450 1. 22-ABC also inhibited the metabolism of benzo(a)pyrene attributable to cytochrome P-450 1 but did not inhibit that induced by treatment with rifampicin or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Analogs of 22-ABC bearing a hydroxyl group or a methylamine in place of the amine moiety exhibit lower affinities for cytochrome P-450 1. In addition, either increasing or decreasing the number of carbons of the side chain reduced the affinity of the inhibitor for cytochrome P-450 1.