title: Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism.
abstract: Sirtuins are protein deacetylases regulating metabolism and stress responses. The seven human Sirtuins (Sirt1-7) are attractive drug targets, but Sirtuin inhibition mechanisms are mostly unidentified. We report the molecular mechanism of Sirtuin inhibition by 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (Ex-527). Inhibitor binding to potently inhibited Sirt1 and Thermotoga maritima Sir2 and to moderately inhibited Sirt3 requires NAD(+), alone or together with acetylpeptide. Crystal structures of several Sirtuin inhibitor complexes show that Ex-527 occupies the nicotinamide site and a neighboring pocket and contacts the ribose of NAD(+) or of the coproduct 2'-O-acetyl-ADP ribose. Complex structures with native alkylimidate and thio-analog support its catalytic relevance and show, together with biochemical assays, that only the coproduct complex is relevant for inhibition by Ex-527, which stabilizes the closed enzyme conformation preventing product release. Ex-527 inhibition thus exploits Sirtuin catalysis, and kinetic isoform differences explain its selectivity. Our results provide insights in Sirtuin catalysis and inhibition with important implications for drug development.
authors.affiliation: Department of Biochemistry and Research Center for Bio-Macromolecules, University of Bayreuth, 95440 Bayreuth, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Gertz
authors.forename: Frank
authors.initials: F
authors.lastname: Fischer
authors.forename: Giang Thi Tuyet
authors.initials: GT
authors.lastname: Nguyen
authors.forename: Mahadevan
authors.initials: M
authors.lastname: Lakshminarasimhan
authors.forename: Mike
authors.initials: M
authors.lastname: Schutkowski
authors.forename: Michael
authors.initials: M
authors.lastname: Weyand
authors.forename: Clemens
authors.initials: C
authors.lastname: Steegborn
mesh_terms: D000107:Acetylation; D002227:Carbazoles / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism*; D037761:Sirtuins / Q000037:antagonists & inhibitors*; D013237:Stereoisomerism
chemical_list: C550547:6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; D002227:Carbazoles; D009243:NAD; D037761:Sirtuins