CAT-2003: A novel sterol regulatory element-binding protein inhibitor that reduces steatohepatitis, plasma lipids, and atherosclerosis in apolipoprotein E*3-Leiden mice.

CAT-2003 is a novel conjugate of eicosapentaenoic acid (EPA) and niacin designed to be hydrolyzed by fatty acid amide hydrolase to release EPA inside cells at the endoplasmic reticulum. In cultured liver cells, CAT-2003 blocked the maturation of sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 proteins and decreased the expression of multiple SREBP target genes, including HMGCR and PCSK9. Consistent with proprotein convertase subtilisin/kexin type 9 (PCSK9) reduction, both low-density lipoprotein receptor protein at the cell surface and low-density lipoprotein particle uptake were increased. In apolipoprotein E*3-Leiden mice fed a cholesterol-containing western diet, CAT-2003 decreased hepatic inflammation and steatosis as evidenced by fewer inflammatory cell aggregates in histopathologic sections, decreased nuclear factor kappa B activity in liver lysates, reduced inflammatory gene expression, reduced intrahepatic cholesteryl ester and triglyceride levels, and decreased liver mass. Plasma PCSK9 was reduced and hepatic low-density lipoprotein receptor protein expression was increased; plasma cholesterol and triglyceride levels were lowered. Aortic root segments showed reduction of several atherosclerotic markers, including lesion size, number, and severity. CAT-2003, when dosed in combination with atorvastatin, further lowered plasma cholesterol levels and decreased hepatic expression of SREBP target genes. Conclusion: SREBP inhibition is a promising new strategy for the prevention and treatment of diseases associated with abnormal lipid metabolism, such as atherosclerosis and nonalcoholic steatohepatitis. (Hepatology Communications 2017;1:311-325).

Synthesis and Characterization of Fatty Acid Conjugates of Niacin and Salicylic Acid.

This report describes the synthesis and preliminary biological characterization of novel fatty acid niacin conjugates and fatty acid salicylate conjugates. These molecular entities were created by covalently linking two bioactive molecules, either niacin or salicylic acid, to an omega-3 fatty acid. This methodology allows the simultaneous intracellular delivery of two bioactives in order to elicit a pharmacological response that could not be replicated by administering the bioactives individually or in combination. The fatty acid niacin conjugate 5 has been shown to be an inhibitor of the sterol regulatory element binding protein (SREBP), a key regulator of cholesterol metabolism proteins such as PCSK9, HMG-CoA reductase, ATP citrate lyase, and NPC1L1. On the other hand, the fatty acid salicylate conjugate 11 has been shown to have a unique anti-inflammatory profile based on its ability to modulate the NF-κB pathway through the intracellular release of the two bioactives.

SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity.

Chronic inflammation is an important etiology underlying obesity-related disorders such as insulin resistance and type 2 diabetes, and recent findings indicate that the macrophage can be the initiating cell type responsible for this chronic inflammatory state. The mammalian silent information regulator 2 homolog SIRT1 modulates several physiological processes important for life span, and a potential role of SIRT1 in the regulation of insulin sensitivity has been shown. However, with respect to inflammation, the role of SIRT1 in regulating the proinflammatory pathway within macrophages is poorly understood. Here, we show that knockdown of SIRT1 in the mouse macrophage RAW264.7 cell line and in intraperitoneal macrophages broadly activates the JNK and IKK inflammatory pathways and increases LPS-stimulated TNFalpha secretion. Moreover, gene expression profiles reveal that SIRT1 knockdown leads to an increase in inflammatory gene expression. We also demonstrate that SIRT1 activators inhibit LPS-stimulated inflammatory pathways, as well as secretion of TNFalpha, in a SIRT1-dependent manner in RAW264.7 cells and in primary intraperitoneal macrophages. Treatment of Zucker fatty rats with a SIRT1 activator leads to greatly improved glucose tolerance, reduced hyperinsulinemia, and enhanced systemic insulin sensitivity during glucose clamp studies. These in vivo insulin-sensitizing effects were accompanied by a reduction in tissue inflammation markers and a decrease in the adipose tissue macrophage proinflammatory state, fully consistent with the in vitro effects of SIRT1 in macrophages. In conclusion, these results define a novel role for SIRT1 as an important regulator of macrophage inflammatory responses in the context of insulin resistance and raise the possibility that targeting of SIRT1 might be a useful strategy for treating the inflammatory component of metabolic diseases.

Crystal structures of human SIRT3 displaying substrate-induced conformational changes.

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.

A protein deacetylase SIRT1 is a negative regulator of metalloproteinase-9.

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.

AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity.

AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the peroxisome proliferator-activated receptor-gamma coactivator 1alpha and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism.

Discovery of oxazolo[4,5-b]pyridines and related heterocyclic analogs as novel SIRT1 activators.

SIRT1 is an NAD(+)-dependent protein deacetylase that appears to produce beneficial effects on metabolic parameters such as glucose and insulin homeostasis. Activation of SIRT1 by resveratrol (1) has been shown to modulate insulin resistance, increase mitochondrial content and prolong survival in lower organisms and in mice on a high fat diet. Herein, we describe the identification and SAR of a series of oxazolo[4,5-b]pyridines as novel small molecule activators of SIRT1 which are structurally unrelated to and more potent than resveratrol.

Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo.

BACKGROUND: Calorie restriction (CR) produces a number of health benefits and ameliorates diseases of aging such as type 2 diabetes. The components of the pathways downstream of CR may provide intervention points for developing therapeutics for treating diseases of aging. The NAD+-dependent protein deacetylase SIRT1 has been implicated as one of the key downstream regulators of CR in yeast, rodents, and humans. Small molecule activators of SIRT1 have been identified that exhibit efficacy in animal models of diseases typically associated with aging including type 2 diabetes. To identify molecular processes induced in the liver of mice treated with two structurally distinct SIRT1 activators, SIRT501 (formulated resveratrol) and SRT1720, for three days, we utilized a systems biology approach and applied Causal Network Modeling (CNM) on gene expression data to elucidate downstream effects of SIRT1 activation. RESULTS: Here we demonstrate that SIRT1 activators recapitulate many of the molecular events downstream of CR in vivo, such as enhancing mitochondrial biogenesis, improving metabolic signaling pathways, and blunting pro-inflammatory pathways in mice fed a high fat, high calorie diet. CONCLUSION: CNM of gene expression data from mice treated with SRT501 or SRT1720 in combination with supporting in vitro and in vivo data demonstrates that SRT501 and SRT1720 produce a signaling profile that mirrors CR, improves glucose and insulin homeostasis, and acts via SIRT1 activation in vivo. Taken together these results are encouraging regarding the use of small molecule activators of SIRT1 for therapeutic intervention into type 2 diabetes, a strategy which is currently being investigated in multiple clinical trials.

Discovery of benzothiazole derivatives as efficacious and enterocyte-specific MTP inhibitors.

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.

Discovery of imidazo[1,2-b]thiazole derivatives as novel SIRT1 activators.

A series of imidazo[1,2-b]thiazole derivatives is shown to activate the NAD(+)-dependent deacetylase SIRT1, a potential new therapeutic target to treat various metabolic disorders. This series of compounds was derived from a high throughput screening hit bearing an oxazolopyridine core. Water-solubilizing groups could be installed conveniently at either the C-2 or C-3 position of the imidazo[1,2-b]thiazole ring. The SIRT1 enzyme activity could be adjusted by modifying the amide portion of these imidazo[1,2-b]thiazole derivatives. The most potent analogue within this series, namely, compound 29, has demonstrated oral antidiabetic activity in the ob/ob mouse model, the diet-induced obesity (DIO) mouse model, and the Zucker fa/fa rat model.

Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3.

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.

SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes.

SIRT1 is a prominent member of a family of NAD(+)-dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.

Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation.

The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly enhances endurance running performance and strongly protects from diet-induced obesity and insulin resistance by enhancing oxidative metabolism in skeletal muscle, liver, and brown adipose tissue. These metabolic effects of SRT1720 are mediated by the induction of a genetic network controlling fatty acid oxidation through a multifaceted mechanism that involves the direct deacetylation of PGC-1alpha, FOXO1, and p53 and the indirect stimulation of AMPK signaling through a global metabolic adaptation mimicking low energy levels. Combined with our previous work on resveratrol, the current study further validates SIRT1 as a target for the treatment of metabolic disorders and characterizes the mechanisms underlying the therapeutic potential of SIRT1 activation.

The Sirtuin family: therapeutic targets to treat diseases of aging.

Sirtuins have emerged as therapeutic targets to treat age-related diseases. There are seven human Sirtuins (SIRT1-7) that display diversity in cellular localization and function. Growing evidence suggests that small-molecule activators of SIRT1 may counteract age-related afflictions such as type 2 diabetes. Alternatively, inhibitors of SIRT2 may be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Recent discoveries of small-molecule and protein modulators of Sirtuin deacetylation activity have provided enormous insight into the biological and molecular functions of Sirtuins and have validated their potential as therapeutics.

Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.

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.