Aromatase inactivation by a suicide substrate, androst-5-ene-4,7,17- trione: The 5β,6β-epoxy-19-oxo derivative, as a possible reactive electrophile irreversibly binding to the active site

Abstract

In order to understand the mechanism involved in the aromatase inactivation by androst-5-ene-4,7,17-trione (4), a suicide substrate of aromatase, 5β,6β-epoxyandrosta-4,7,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase upon treatment of 19-oxo-5-ene steroid 5 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 was a competitive inhibitor of human placental aromatase (K(i) = 34 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K(i) = 36 μM, a rate constant for inactivation (k(inact)) = 0.027 min-1). NADPH stimulated the inactivation rate, but the substrate androst- 4-ene-3,17-dione blocked the inactivation. A nucleophile, L-cysteine, did not cause a significant change in the inactivation. When both the epoxide 6 and its 19-methyl analog 7 were subjected separately to a reaction with N- acetyl-L-cysteine in the presence of NaHCO3, the 19-oxo compound 6 disappeared from the reaction mixture more rapidly (t( 1/4 )=6.0 min) than the 19-methyl analog 7 (t( 1/4 )= 16 min). On the basis of these results, it is suggested that the 5β,6β-epoxy-19-oxo steroid 6 may be the reactive electrophile that alkylates a nucleophilic residue of the amino acid of the active site.