title: Inhibition of human steroid 5alpha reductases type I and II by 6-aza-steroids: structural determinants of one-step vs two-step mechanism.
abstract: We have discovered that 17beta-[N,N-(diethyl)carbamoyl]-6-azaandrost-4-en-3-one is a time-dependent inhibitor of type II 5alpha-reductase, as is the drug finasteride. Unlike finasteride, the 6-aza-steroid is not a time-dependent inhibitor of type I 5 alpha-reductase. Finasteride inhibition of type II enzyme proceeds in a two-step mechanism. At pH 6 and 37 degrees C, an initial finasteride-reductase complex is formed with a K(i)(app) of 11.9 +/- 4.1 nM. In a second step, an irreversible complex is formed with a rate constant of inactivation of 0.09 +/- 0.01 s(-1). In contrast, the 6-aza-steroid is a reversible inhibitor. From the results of a simplified mathematical analysis, based on the rapid equilibrium approximation, the inhibitor and the enzyme form an initial complex with a K(i) of 6.8 +/- 0.2 nM. The reversible formation of a final complex, with an overall K(i) of 0.07 +/- 0.02 nM, is characterized by a first-order isomerization rate constant 0.0035 +/- 0.0001 s(-1) for the forward step and 0.00025 +/- 0.00006 s(-1) for the backward step. All rate constants for the two-step mechanism were obtained by using a general numerical integration method. The best fit values for the association and dissociation rate constants were 5.0 microM(-1) s(-1) and 0.033 +/- 0.008 s(-1), respectively, and the isomerization rate constants were 0.0035 +/- 0.007 s(-1) and 0.000076 +/- 0.000019 s(-1). These values correspond to an initial K(i) of 6.5 nM and an overall dissociation constant of 0.14 nM. The data presented here show that both finasteride and the 6-aza-steroid analogs are potent against type II 5alpha-reductase, although their mechanisms of inhibition are different.
authors.affiliation: Department of Enzymology, Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Moss
authors.forename: P
authors.initials: P
authors.lastname: Kuzmic
authors.forename: J D
authors.initials: JD
authors.lastname: Stuart
authors.forename: G
authors.initials: G
authors.lastname: Tian
authors.forename: A G
authors.initials: AG
authors.lastname: Peranteau
authors.forename: S V
authors.initials: SV
authors.lastname: Frye
authors.forename: S H
authors.initials: SH
authors.lastname: Kadwell
authors.forename: T A
authors.initials: TA
authors.lastname: Kost
authors.forename: L K
authors.initials: LK
authors.lastname: Overton
authors.forename: I R
authors.initials: IR
authors.lastname: Patel
mesh_terms: D058891:5-alpha Reductase Inhibitors; D001378:Azasteroids / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D018120:Finasteride / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D013329:Structure-Activity Relationship
chemical_list: D058891:5-alpha Reductase Inhibitors; D001378:Azasteroids; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D018120:Finasteride