Design and synthesis of amino acid derivatives of substituted benzimidazoles and pyrazoles as Sirt1 inhibitors.

Owing to its presence in several biological processes, Sirt1 acts as a potential therapeutic target for many diseases. Here, we report the structure-based designing and synthesis of two distinct series of novel Sirt1 inhibitors, benzimidazole mono-peptides and amino-acid derived 5-pyrazolyl methylidene rhodanine carboxylic acid. The compounds were evaluated for in vitro enzyme-based and cell-based Sirt1 inhibition assay, and cytotoxic-activity in both liver and breast cancer cells. The tryptophan conjugates i.e.13h (IC50 = 0.66 µM, ΔG bind = -1.1 kcal mol-1) and 7d (IC50 = 0.77 µM, ΔG bind = -4.4 kcal mol-1) demonstrated the maximum efficacy to inhibit Sirt1. The MD simulation unveiled that electrostatic complementarity at the substrate-binding-site through a novel motif "SLxVxP(V/F)A" could be a cause of increased Sirt1 inhibition by 13h and 13l over Sirt2 in cell-based assay, as compared to the control Ex527 and 7d. Finally, this study highlights novel molecules 7d and 13h, along with a new key hot-spot in Sirt1, which could be used as a starting lead to design more potent and selective sirtuin inhibitors as a potential anticancer molecule.

Sirt1 and Sirt3 Activation Improved Cardiac Function of Diabetic Rats via Modulation of Mitochondrial Function.

In the present study, we aimed to evaluate the effect of Sirt1, Sirt3 and combined activation in high fructose diet-induced insulin resistance rat heart and assessed the cardiac function focusing on mitochondrial health and function. We administered the Sirt1 activator; SRT1720 (5 mg/kg, i.p.), Sirt3 activator; Oroxylin-A (10 mg/kg i.p.) and the combination; SRT1720 + Oroxylin-A (5 mg/kg and 10 mg/kg i.p.) daily from 12th week to 20th weeks of study. We observed significant perturbations of most of the cardiac structural and functional parameters in high fructose diet-fed animals. Administration of SRT1720 and Oroxylin-A improved perturbed cardiac structural and functional parameters by decreasing insulin resistance, oxidative stress, and improving mitochondrial function by enhancing mitochondrial biogenesis, OXPHOS expression and activity in high fructose diet-induced insulin-resistant rats. However, we could not observe the synergistic effect of SRT1720 and Oroxylin-A combination. Similar to in-vivo study, perturbed mitochondrial function and oxidative stress observed in insulin-resistant H9c2 cells were improved after activation of Sirt1 and Sirt3. We observed that Sirt1 activation enhances Sirt3 expression and mitochondrial biogenesis, and the opposite effects were observed after Sirt1 inhibition in cardiomyoblast cells. Taken together our results conclude that activation of Sirt1 alone could be a potential therapeutic target for diabetes-associated cardiovascular complications.

Allyl Methyl Sulfide Preserved Pressure Overload-Induced Heart Failure Via Modulation of Mitochondrial Function.

BACKGROUND: Cardiovascular diseases are the leading cause of death globally, and they are causing enormous socio-economic burden to the developed and developing countries. Allyl Methyl Sulfide (AMS) is a novel cardioprotective metabolite identified in the serum of rats after raw garlic administration. The present study explored the cardioprotective effect of AMS on thoracic aortic constriction (TAC)-induced cardiac hypertrophy and heart failure model in rats. METHODS: Thoracic aortic constriction (TAC) by titanium ligating clips resulted in the development of pressure overload-induced cardiac hypertrophy and heart failure model. Four weeks prior to TAC and for 8 weeks after TAC, Sprague Dawley (SD) rats were administered with AMS (25 and 50 mg/kg/day) or Enalapril (10 mg/kg/day). RESULTS: We have observed AMS (25 and 50 mg/kg/day) intervention significantly improved structural and functional parameters of the heart. mRNA expression of fetal genes i.e., atrial natriuretic peptide (ANP), alpha skeletal actin (α-SA) and beta myosin heavy chain (ß-MHC) were reduced in AMS treated TAC hearts along with decrease in perivascular and interstitial fibrosis. AMS attenuated lipid peroxidation and improved protein expression of endogenous antioxidant enzymes i.e., catalase and manganese superoxide dismutase (MnSOD) along with electron transport chain (ETC) complex activity. AMS increased mitochondrial fusion proteins i.e., mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy protein (OPA1), and reduced fission protein i.e., dynamin-related protein 1 (DRP1). Preliminary study suggests that AMS intervention upregulated genes involved in mitochondrial bioenergetics in normal rats. Further, in-vitro studies suggest that AMS reduced mitochondrial reactive oxygen species (ROS), preserved mitochondrial membrane potential and oxygen consumption rate (OCR) in isoproterenol-treated cardiomyoblast. CONCLUSION: This study demonstrated that AMS protected cardiac remodelling, LV dysfunction and fibrosis in pressure overload-induced cardiac hypertrophy and heart failure model by improving endogenous antioxidants and mitochondrial function.

Activation of toll like receptor 4 (TLR4) promotes cardiomyocyte apoptosis through SIRT2 dependent p53 deacetylation.

Cardiomyocyte inflammation followed by apoptosis and fibrosis is an important mediator for development and progression of heart failure. Activation of toll-like receptor 4 (TLR4), an important regulator of inflammation, causes the progression of cardiac hypertrophy and injury. However, the precise mechanism of TLR4-mediated adverse cardiac outcomes is still elusive. The present study was designed to find the role of TLR4 in cardiac fibrosis and apoptosis, and molecular mechanism thereof. Rats were treated with TLR4 agonist (LPS 12.5 µg/kg/day) through osmotic pump for 14 days. To simulate the condition in vitro, H9c2 cells were treated with LPS (1 µg/ml). Similarly, H9c2 cells were transfected with TLR4 and SIRT2 c-DNA clone for overexpression. Myocardial oxidative stress, inflammation, fibrosis and mitochondrial parameters were evaluated both in vitro and in vivo. Cardiac inflammation after LPS treatment was confirmed by increased TNF-α and IL-6 expression in rat heart. There was a marked increase in oxidative stress as observed by increased TBARS and decreased endogenous antioxidants (GSH and catalase), along with mitochondrial dysfunction as measured by mitochondrial complex activity in LPS-treated rat hearts. Histopathological examination showed the presence of cardiac fibrosis after LPS treatment. Protein expression of nuclear p53 and cleaved caspase-7/caspase-9 was significantly increased in LPS treated heart. Similar to in vivo study, nuclear translocation of p53, mitochondrial dysfunction and cellular apoptosis were observed in H9c2 cells treated with LPS. Our data also indicate that decreased expression of SIRT2 was associated with increased acetylation of p53 after LPS treatment. In conclusion, TLR4 activation in rats promotes cardiac inflammation, mitochondrial dysfunction, apoptosis and fibrosis. p53 and caspase 7/caspase 9 were found to play an important role in TLR4-mediated apoptosis. Our data suggest that, reducing TLR4 mediated fibrosis and apoptosis could be a novel approach in the treatment of heart failure, keeping in the view the major role played by TLR4 in cardiac inflammation.

Allylmethylsulfide, a Sulfur Compound Derived from Garlic, Attenuates Isoproterenol-Induced Cardiac Hypertrophy in Rats.

Allylmethylsulfide (AMS) is a novel sulfur metabolite found in the garlic-fed serum of humans and animals. In the present study, we have observed that AMS is safe on chronic administration and has a potential antihypertrophic effect. Chronic administration of AMS for 30 days did not cause any significant differences in the body weight, electrocardiogram, food intake, serum biochemical parameters, and histopathology of vital organs. Single-dose pharmacokinetics of AMS suggests that AMS is rapidly metabolized into Allylmethylsulfoxide (AMSO) and Allylmethylsulfone (AMSO2). To evaluate the efficacy of AMS, cardiac hypertrophy was induced by subcutaneous implantation of ALZET® osmotic minipump containing isoproterenol (~5 mg/kg/day), cotreated with AMS (25 and 50 mg/kg/day) and enalapril (10 mg/kg/day) for 2 weeks. AMS and enalapril significantly reduced cardiac hypertrophy as studied by the heart weight to body weight ratio and mRNA expression of fetal genes (ANP and ß-MHC). We have observed that TBARS, a parameter of lipid peroxidation, was reduced and the antioxidant enzymes (glutathione, catalase, and superoxide dismutase) were improved in the AMS and enalapril-cotreated hypertrophic hearts. The extracellular matrix (ECM) components such as matrix metalloproteinases (MMP2 and MMP9) were significantly upregulated in the diseased hearts; however, with the AMS and enalapril, it was preserved. Similarly, caspases 3, 7, and 9 were upregulated in hypertrophic hearts, and with the AMS and enalapril treatment, they were reduced. Further to corroborate this finding with in vitro data, we have checked the nuclear expression of caspase 3/7 in the H9c2 cells treated with isoproterenol and observed that AMS cotreatment reduced it significantly. Histopathological investigation of myocardium suggests AMS and enalapril treatment reduced fibrosis in hypertrophied hearts. Based on our experimental results, we conclude that AMS, an active metabolite of garlic, could reduce isoproterenol-induced cardiac hypertrophy by reducing oxidative stress, apoptosis, and stabilizing ECM components.

Musa balbisiana Fruit Rich in Polyphenols Attenuates Isoproterenol-Induced Cardiac Hypertrophy in Rats via Inhibition of Inflammation and Oxidative Stress.

Musa balbisiana Colla (Family: Musaceae), commonly known as banana and native to India and other parts of Asia, is very rich in nutritional value and has strong antioxidant potential. In the present study, we have developed Musa balbisiana (MB) fruit pulp powder and evaluated its cardioprotective effect in cardiac hypertrophy, which is often associated with inflammation and oxidative stress. An ultra-high-pressure liquid chromatography-mass spectrometer (UPLC-MS/MS) has been used for the detection and systematic characterization of the phenolic compounds present in Musa balbisiana fruit pulp. The cardioprotective effect of MB was evaluated in a rat model of isoproterenol- (ISO-) induced cardiac hypertrophy by subcutaneous administration of isoproterenol (5 mg/kg-1/day-1), delivered through an alzet minipump for 14 days. Oral administration of MB fruit pulp powder (200 mg/kg/day) significantly (p < 0.001) decreased heart weight/tail length ratio and cardiac hypertrophy markers like ANP, BNP, ß-MHC, and collagen-1 gene expression. MB also attenuated ISO-induced cardiac inflammation and oxidative stress. The in vivo data were further confirmed in vitro in H9c2 cells where the antihypertrophic and anti-inflammatory effect of the aqueous extract of MB was observed in the presence of ISO and lipopolysaccharide (LPS), respectively. This study strongly suggests that supplementation of dried Musa balbisiana fruit powder can be useful for the prevention of cardiac hypertrophy via the inhibition of inflammation and oxidative stress.