Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu230, located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...

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SIRT3 is a major mitochondrial NAD؉ -dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD ؉ . In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD ؉ . These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.Sirtuins are class III histone deacetylases that couple lysine deacetylation with NAD ϩ hydrolysis and are highly conserved in prokaryotes and eukaryotes (1). Mammals possess seven sirtuins, SIRT1-7, that occupy different subcellular compartments such as the nucleus (SIRT1, -6, -7), cytoplasm (SIRT2), and the mitochondria (SIRT3, -4, and -5) (2). They deacetylate lysines not only on histone substrates (3, 4) but also on nonhistone substrates such as p53 tumor suppressor protein (5), Foxo transcription factors (6, 7), PGC-1␣ (8), ␣-tubulin (9), acetyl-CoA synthetases (10 -12), and glutamate dehydrogenase (13). SIRT4 and SIRT6 have been shown to have ADP-ribosyltransferase activity (14 -16). Sirtuins have been reported to play important roles in gene silencing (17), cell cycle regulation (18,19), metabolism (8, 10 -12, 14, 20 -22), apoptosis (5, 23, 24), the lifespan-extension effects of calorie restriction (25,26), and circadian rhythms (27)(28)(29)(30) (50), and SIRT5 (51). Sirtuins contain a conserved enzymatic core with two domains; that is, a large Rossmann fold domain that binds NAD ϩ and a small domain formed by two insertions of the large domain that binds to a zinc atom. The acetylated peptide substrate binds to the cleft between the two domains. Some of the known structures are apo structures with sirtuin protein alone, whereas others are bound to acetylated peptide substrate and/or NAD ϩ and its analogs. These structures revealed the mechanism of action for the deacetylation activity and substrate specificity.SIRT3 localizes in mitochondria (13, 52-54) and is a major mitochondrial deacetylase. Hyperacetylation of mitochondrial proteins have been observed in SIRT3 knock-out mice (13, 55). Several key enzymes involved in energy production in the mitochondria have been identified as SIRT3 substrates. Acetyl-CoA synthetase 2 (AceCS2) 2 converts acetate into acetyl-CoA in the mitochondria. Deacetyla...