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A series of triamide derivatives bearing a benzothiazole core is shown to be potent microsomal triglyceride transfer protein (MTP) inhibitors. In order to minimize liver toxicity, these compounds have been optimized to have activity only in the enterocytes and have limited systemic bioavailability. Upon oral administration, selected analogs within this series have been further demonstrated to reduce food intake along with body weight and thereby improve glucose homeostasis and insulin sensitivity in a 28-day mice diet-induced obesity (DIO) model.

SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C-terminus of human SIRT3 but lacks an N-terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5′ rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full-length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD+. Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes1,2. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity3–9. 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 survival10–14. 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.

Fabry disease is an X-linked recessive disorder in which affected persons lack alpha-galactosidase A (alpha -GalA), which leads to excess glycosphingolipids in tissues, mainly globotriaosylceramide (Gb3). Gb3 is the cellular receptor for Shiga toxin (Stx), the primary virulence factor of enterohemorrhagic Escherichia coli. alpha-GalA-knockout mice were significantly protected against lethal intraperitoneal doses of Stx2 or oral doses of Stx2-expressing bacteria, compared with wild-type (wt) control mice. Kidneys of moribund wt mice revealed tubular necrosis, but no histopathologic changes were observed in Gb3-overexpressing mice. Reducing Gb3 levels in alpha-GalA-knockout mice by the intravenous injection of recombinant human alpha-GalA restored the susceptibility of knockout mice to lethal doses of Stx2. These results suggest that excess amounts of Gb3 in alpha-GalA-deficient mice may impair toxin delivery to susceptible tissues.

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.

In this report, we describe the use of an attenuated regulatory mutant of a rabbit enteropathogenic Escherichia coli (rEPEC) as a live vaccine vector to deliver heterologous protein antigens using two dedicated transport systems, a Salmonella autotransporter and the E. coli hemolysin apparatus. We previously reported that an isogeneic ler (LEE encoded regulator) mutant of rEPEC O103:H2 is attenuated and immunogenic in rabbits. We first evaluated the Salmonella autotransporter MisL containing the immunodominant B-cell epitope of the circumsporozoite protein from Plasmodium falciparum, (NANP)8, fused to the C-terminal translocator domain under the control of the constitutive Tac17 promoter. The rEPEC ler mutant was able to express and to translocate the (NANP)8 passenger peptide to the bacterial surface. We next investigated the delivery of Shiga toxin B subunit (Stx1B) from human enterohemorrhagic E. coli by the rEPEC ler mutant via the MisL autotransporter or the E. coli hemolysin secretion apparatus. The autotransporter and hemolysin plasmids expressed similar levels of Stx1B (30-40 ng/ml/OD600). Only 6% of Stx1B was found in the autotransporter supernatants; the rest was cell-associated, with a small fraction of the Stx1B surface-exposed as determined by immunofluorescence. In contrast, 88% of Stx1B was secreted into culture supernatants by the hemolysin secretion system. In an in vivo study, no significant protection was observed in rabbits inoculated with the ler mutant harboring the Stx1B-autotransporter plasmid following experimental challenge with RDEC-H19A, the prototype rEPEC containing an Stx-converting phage. In contrast, rabbits inoculated with the rEPEC ler mutant containing the Stx1B-hemolysin fusion were partially protected from RDEC-H19A infection as demonstrated by decreased weight loss (p