Aromatase inactivation by a suicide substrate, androst-5-ene-4,7,17-trione: the 5beta,6beta-epoxy-19-oxo derivative, as a possible reactive electrophile irreversibly binding to the active site.
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, 5beta,6beta-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 (Ki = 34 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (Ki = 36 microM, 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 (t1/2 = 6.0 min) than the 19-methyl analog 7 (t1/2 = 16 min). On the basis of these results, it is suggested that the 5beta,6beta-epoxy-19-oxo steroid 6 may be the reactive electrophile that alkylates a nucleophilic residue of the amino acid of the active site.
Numazawa
M
M
Tohoku College of Pharmacy, Aoba-ku, Sendai, Japan.
Tachibana
M
M
D000111:Acetylcysteine / Q000737:chemistry; D000731:Androstanes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000735:Androstenedione / Q000494:pharmacology; D000736:Androstenes / Q000378:metabolism / Q000494:pharmacology*; D047072:Aromatase Inhibitors; D001665:Binding Sites; D001667:Binding, Competitive; D003545:Cysteine / Q000494:pharmacology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
C106912:5,6-epoxyandrosta-4,7,17,19-tetraone; C106913:5,6-epoxyandrosta-4,7,17-trione; D000731:Androstanes; D000736:Androstenes; D047072:Aromatase Inhibitors; C106911:androst-5-en-4,7,17-trione; D000735:Androstenedione; D009249:NADP; D003545:Cysteine; D000111:Acetylcysteine