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. 2013 Jul 23;110(30):E2772-81.
doi: 10.1073/pnas.1303628110. Epub 2013 Jul 9.

Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism

Melanie Gertz  1 , Frank Fischer, Giang Thi Tuyet Nguyen, Mahadevan Lakshminarasimhan, Mike Schutkowski, Michael Weyand, Clemens Steegborn
Affiliations

Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism

Melanie Gertz et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Potency and kinetics of Sirtuin inhibition by Ex-527. (A) Mechanism proposed for Sirtuin-dependent deacetylation, illustrated with crystal structures: apo-enzyme (Sirt3 3GLS) (13), substrate complex (Sir2Tm 2H4F) (16), alkylimidate intermediate (Sirt3 8GLT) (13), bicyclic intermediate (Sirt5 4F56) (57), and product complex (Hst2 1Q1A) (14). (B) Chemical structure of Ex-527, with ring systems labeled A to C (*stereocenter). (C) Ex-527 dose–response experiments for Sirt1, Sirt3, and Sir2Tm (fitted with a tight-binding equation). (D) Sirt5 deacetylation and desuccinylation activity in the presence and absence of Ex-527. (E and F) Peptide substrate-dependent activity of Sir2Tm (E) and Sirt3 (F) in the absence and presence of varying Ex-527 concentrations revealing noncompetitive inhibition. (G and H) NAD+-dependent activity of Sir2Tm (G) and Sirt3 (H) at varying Ex-527 concentrations revealing uncompetitive inhibition. (D, E, and G) Error bars represent SEs of linear fits to time series experiments. (C, F, and H) Regression coefficients of linear fits to time series experiments were >0.95.
Fig. 2.
Fig. 2.
Crystal structures of Ex-243 complexes with Sir2Tm and Sirt3. (A) Overall structures of Sir2Tm/Ex-243 complex (brown), Sirt3/NAD+/Ex-243 (green), and apo Sirt3 (PDB ID 3GLS, blue) (13). (B) Active site of the Sir2Tm structure obtained through NAD+/Ex-527 soaking and enzymatic turnover resulting in Sir2Tm/substrates and Sir2Tm/products/Ex-243 complexes. (C) Sigma-A–weighted Fo-Fc density for products and Ex-243 contoured at 2.5 σ. (D) Structure of the Sir2Tm/products/Ex-243 complex obtained by cocrystallization. (E) Schematic view of the interactions between Ex-243 and Sir2Tm. (F) Structure of Sirt3 in complex with NAD+ in elongated and nonproductive conformation and Ex-243 bound to the C-pocket. (G) Structure of the Sirt3/ADP ribose/Ex-243 complex. (A–G) Proteins are shown in cartoon representation; ligands as sticks colored by atom type. (D, F, and G) The sigma-A–weighted 2Fo-Fc maps are contoured at 1.0 σ.
Fig. 3.
Fig. 3.
Coligand for the inhibitory Ex-243 complex. (A and B) Thermophoresis binding experiments to determine Ex-243 affinities to different states of Sir2Tm (A) and Sirt3 (B). Error bars represent SEs of two independent measurements. (C–E) Product formation quantified by MS at various Sirt3 (C), Sirt1 (D), and Sir2Tm (E) concentrations in the presence or absence of Ex-243 and Ro-31-8220. The dashed line indicates the formation of one product molecule per Sirtuin molecule. Error bars represent SEs of two independent measurements. The control in E is saturated. (F) Structure of Sirt3 in complex with native alkylimidate intermediate. The sigma-A–weighted 2Fo-Fc map is contoured at 1.0 σ. (G) Overlay of Sirt3 bound to native alkylimidate intermediate or its thio-analog. Ex-243 was modeled into the C-site by overlaying the structures with the Sirt3/NAD+/Ex-243 complex revealing clashes between intermediate and Ex-243 (dashed line). (F and G) Proteins are shown in cartoon representation; ligands as sticks colored by atom type. rel. thermophoresis, relative thermophoresis; deac. peptide, decaetylated peptide.
Fig. 4.
Fig. 4.
Structural mechanism of Ex-243 inhibition and isoform selectivity. (A) Structure of Sirt3 bound to 2′-O-acetyl ADP ribose and Ex-243. The sigma-A–weighted 2Fo-Fc map is contoured at 1.0 σ. (B) Comparison of the cofactor binding loop in the Sirt3/2′-O-acetyl ADP ribose/Ex-243 complex (green) with apo Sirt3 (PDB ID 3GLS, blue) (13) and Sirt3 in complex with thio-intermediate (this study, orange). Ex-243 and 2′-O-acetyl-ADP ribose are from the respective Sirt3 complex structure; ligands from the other structures are omitted for clarity. The loop’s movement and the rotation of the Phe157 side chain are indicated by arrows. (A and B) Proteins are shown in cartoon representation; ligands and residues involved in Ex-243 binding as sticks colored by atom type. (C) kcat/KM ratios obtained from NAD+ titration experiments with Sirt3 and Sir2Tm in the presence and absence of Ex-243. (D) Scheme describing the mechanism of Sirtuin inhibition by Ex-243.
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References

    1. Michan S, Sinclair D. Sirtuins in mammals: Insights into their biological function. Biochem J. 2007;404(1):1–13. - PMC - PubMed
    1. Haigis MC, Sinclair DA. Mammalian sirtuins: Biological insights and disease relevance. Annu Rev Pathol. 2010;5:253–295. - PMC - PubMed
    1. Sauve AA, Wolberger C, Schramm VL, Boeke JD. The biochemistry of sirtuins. Annu Rev Biochem. 2006;75:435–465. - PubMed
    1. Gertz M, Steegborn C. Function and regulation of the mitochondrial sirtuin isoform Sirt5 in Mammalia. Biochim Biophys Acta. 2010;1804(8):1658–1665. - PubMed
    1. Bell EL, Guarente L. The SirT3 divining rod points to oxidative stress. Mol Cell. 2011;42(5):561–568. - PMC - PubMed

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