The sirtuin (silent information regulator 2) proteins are NAD + -dependent deacetylases that are implicated in diverse biological processes including DNA regulation, metabolism and longevity. Homologues of the prototypic yeast Sir2p have been identified in all three kingdoms of life, and while bacteria and archaea typically contain one to two sirtuins, eukaryotic organisms contain multiple members. Sirtuins are regulated in part by the cellular concentrations of the noncompetitive inhibitor, nicotinamide, and several synthetic modulators of these enzymes have been identified. The x-ray crystal structures of several sirtuin proteins in various liganded forms have been determined. This wealth of structural information, together with related biochemical studies, have provided important insights into the catalytic mechanism, substrate specificity, and inhibitory mechanism of sirtuin proteins. Implications for future structural studies to address outstanding questions in the field are also discussed.

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, 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 Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

The sirtuin proteins are broadly conserved NAD + -dependant deacetylases that are implicated in diverse biological processes including DNA recombination and repair, transcriptional silencing, longevity, apoptosis, axonal protection, insulin signaling and fat mobilization. Because of these associations, the identification of small molecule sirtuin modulators has been of significant interest. Here we report on high throughput screening against the yeast sirtuin, Hst2, leading to the identification of four unique inhibitor scaffolds that also inhibit the human sirtuins, SIRT1, SIRT2 and SIRT3. The identified inhibitor scaffolds range in potency from IC 50 values of 6.5-130 μ M against Hst2. Each of the inhibitor scaffolds binds reversibly to the enzyme, and kinetic analysis reveals that each of the inhibitors is non-competitive with respect to both acetyl-lysine and NAD + binding. Limited SAR analysis of the scaffolds also identifies which functional groups may be important for inhibition. These sirtuin inhibitors are low molecular weight and well-suited for lead molecule optimization, making them useful chemical probes to study the mechanism and biological roles of sirtuins and potential starting points for optimization into therapeutics.