MP62-08 THE CATALYTIC SITE COMMON TO FOUR 3?-OXIDOREDUCTASES CONTRIBUTES TO INTRACRINE SYNTHESIS OF DHT

Abstract

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2016MP62-08 THE CATALYTIC SITE COMMON TO FOUR 3?-OXIDOREDUCTASES CONTRIBUTES TO INTRACRINE SYNTHESIS OF DHT Michael Fiandalo, John Wilton, John Stocking, Krystin Mongiardo, Elena Pop, Yun Li, Kris Attwood, Elisabeth Wilson, and James Mohler Michael FiandaloMichael Fiandalo More articles by this author , John WiltonJohn Wilton More articles by this author , John StockingJohn Stocking More articles by this author , Krystin MongiardoKrystin Mongiardo More articles by this author , Elena PopElena Pop More articles by this author , Yun LiYun Li More articles by this author , Kris AttwoodKris Attwood More articles by this author , Elisabeth WilsonElisabeth Wilson More articles by this author , and James MohlerJames Mohler More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.916AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. One mechanism that may contribute to CaP resistance to ADT is intracrine metabolism, the conversion of weak adrenal androgens to testicular androgens, testosterone (T) or dihydrotestosterone (DHT). Previous work from our laboratory has shown that the primary backdoor pathway generates DHT without using T as an intermediate substrate. The final step in the primary backdoor pathway involves the conversion of 5-androstane3α,17β-diol (androstanediol) to DHT by 4 enzymes, HSD17B6, RDH16, DHRS9 or RDH5. Inhibition of the step immediately proximate to intracrine metabolism of DHT should lower DHT more effectively than inhibitors of 5α-reductases and/or CYP17A1, which act earlier in intracrine metabolism pathways. The goal of this project is to inhibit the enzymes responsible for the conversion of androstanediol to DHT. METHODS Bioinformatics tools were used to analyze the protein sequences of these 4 enzymes to determine if these enzymes shared common catalytic amino acid residues. Site-directed mutagenesis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to test the catalytic significance of the common catalytic amino acid residues and if mutation of these catalytic residues inhibited androstanediol conversion to DHT. RESULTS The NCBI Constraint-based Multiple Protein Alignment Tool (COBALT) showed that the 4 enzymes share catalytic amino acid residues. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that these 4 enzymes convert androstanediol to DHT and androsterone to androstanedione. Site-directed mutagenesis was used to generate single, double or catalytic deletion enzyme mutants. LC-MS/MS showed that single, double and complete catalytic deletion of the common catalytic amino acids impaired enzyme activity. Furthermore, the impaired catalytic mutants combined with dutasteride decreased CaP cell line DHT levels more than dutasteride alone. CONCLUSIONS These results demonstrate that a new treatment directed against the terminal steps in the primary backdoor pathway may decrease DHT levels better than ADT alone. Further reduction of tissue DHT levels by inhibiting the last step in intracrine metabolism may improve response to ADT or induce re-remission of castration-recurrent CaP (CRCP) and improve survival of men with advanced CaP. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e814-e815 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Michael Fiandalo More articles by this author John Wilton More articles by this author John Stocking More articles by this author Krystin Mongiardo More articles by this author Elena Pop More articles by this author Yun Li More articles by this author Kris Attwood More articles by this author Elisabeth Wilson More articles by this author James Mohler More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...