Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane.
The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.
Division of Basic Medical Sciences, Mercer University School of Medicine, 1550 College Street, Macon, GA 31207, USA. Thomas.J@mercer.edu
James L
JL
Thomas
Vance L
VL
Mack
Jason A
JA
Glow
Delaram
D
Moshkelani
J Ross
JR
Terrell
Kevin M
KM
Bucholtz
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013739:Testosterone / Q000031:analogs & derivatives / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism
D007527:Isoenzymes; D013196:Dihydrotestosterone; D013912:Threonine; D013739:Testosterone; D008715:Methionine; D015096:3-Hydroxysteroid Dehydrogenases; D013251:Steroid Isomerases; C009954:trilostane