Structural and biochemical insights into the mechanism of sirtuin catalysis and regulation by nicotinamide and small molecule inhibitors

Reversible lysine acetylation is a mechanism organisms use to regulate essential cellular activities including gene expression, apoptosis and stress responses. Silent information regulator 2 (Sir2) or sirtuin proteins are broadly conserved NAD+-dependent deacetylases that regulate chromatin function through deacetylation of histone tail lysine residues. Additionally, sirtuin proteins deacetylate many non-histone proteins implicating them in diverse biological processes including longevity, metabolism, axonal protection, insulin signaling and fat mobilization. The NAD+-dependence of this protein family along with the identification of sirtuin proteins as limiting determinants of lifespan in several organisms has proved to be the link in the longstanding association between nutrient-sensing and longevity. Sirtuin proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a sirtuin reaction intermediate via a base exchange reaction to reform NAD+ at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in sirtuin enzymes, we discuss in Chapter 2 the structure of nicotinamide bound to a Sir2 homologue, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base exchange site that is distinct from the so-called C pocket binding site for the nicotinamide group of NAD+. These results provide insights into the sirtuin mechanism of nicotinamide inhibition and have important implications for the development of sirtuin-specific effectors. Because of the many cellular roles sirtuin proteins play, some with implications in human disease, the identification of small molecule sirtuin modulators has been of significant interest. In Chapter 3, we report a high throughput screen against the yeast sirtuin, Hst2, leading to the identification of six unique inhibitor scaffolds that also inhibit the human sirtuins, SIRT1-3, and are able to inhibit telomeric silencing of yeast Sir2 in vivo . The identified inhibitor scaffolds are among the most potent known sirtuin effectors, and each has been characterized with respect to its reversibility of enzyme binding, inhibition mode and binding affinity. Further, limited SAR analysis of the scaffolds also identifies which functional groups may be important for inhibition. These sirtuin inhibitors represent useful chemical probes to study the mechanism and biological roles of sirtuins and potential starting points for optimization into therapeutics. Chapter 4 describes the purification and biochemical characterization of the human sirtuins SIRT2 and SIRT6, along with the pursuit of crystals for their structure determination by X-ray diffraction