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

Abstract

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.

Figure 1. Identification of potent SIRT1 activators unrelated to resveratrol

a, Chemical structures of SIRT1 activators, resveratrol, SRT1460, SRT2183, and SRT1720. b, The effect of activators on human SIRT1 enzyme activity measured by mass spectrometry. c, Cellular activity was measured using an ICW that monitors the degree of p53 deacetylation in U2OS cells using β-tubulin as a normalization control. Compounds were tested at concentrations of: resveratrol (100 μM), SRT2183 (10 μM), SRT1460 (10 μM), SRT1720 (0.10 μM). Each concentration represents the approximate EC₅₀ for each compound. n = 6 for all compounds tested except for resveratrol, where n = 3. Data are expressed as mean ± s.d. The activation of SIRT1 resulting in p53 deacetylation could be blocked by a SIRT1 inhibitor, 6-chloro-2,3,4,9-tetrahydro-1-H-carbazole-1-carboxamide (10 μM). n = 3 for all compounds tested.

Figure 2. In vitro characterization of activators of human SIRT1

a, The effect of SIRT1 activators on peptide substrate K_m. b, Calorimetric titrations of SIRT1-C—peptide substrate complex with the activator SRT1460. Top panel: heat of binding SRT1460 to enzyme—peptide complex. Bottom panel: integrated fit with a one-site binding model. c, Isobologram analysis of resveratrol versus SRT1720 and SRT1720 versus SRT1460. The experimental data are best fit to the theoretical line of additivity (dashed line). d, SIRT1 N-terminal truncations define the allosteric compound binding site. The ability of resveratrol and SRT1720 to activate SIRT1 was examined against a series of N-terminal deletions in the mass spectrometry assay.

Figure 3. SIRT1 activators in mouse models of type 2 diabetes

a, Plasma levels of SRT1720 after administration by oral gavage. b, Effect of SRT1720 treatment over 10 weeks on fed plasma glucose levels in DIO mice. c, Glucose excursion during an intraperitoneal glucose tolerance test (5 weeks) in DIO mice treated with the indicated compounds. d, Plasma insulin levels after 10 weeks treatment with indicated compounds. e, Glucose response in DIO mice during an insulin tolerance test after 10 weeks treatment with the indicated compounds. f, Skeletal muscle citrate synthase activity (V_maxmg⁻¹ protein, 11 weeks, n = 5). g, Effect of SRT1720 treatment in diabetic Lep^ob/ob mice (1 week). h, Effect of SRT501 treatment (1,000 mg per kg (body weight)) in diabetic Lep^ob/ob mice after 2 weeks. i, Effect of SRT501 treatment (500 mg per kg (body weight), 4 weeks) in DIO mice. All studies consisted of ten mice per group unless noted. Statistics were conducted as an ANOVA; asterisk P < 0.05, double asterisk P < 0.01 and triple asterisk P < 0.001. Data are expressed as mean ± s.e.m.

Figure 4. SIRT1 activator SRT1720 in the Zucker fa/fa rat model

a, Post-absorptive blood glucose (3 weeks). b, c, Glucose and insulin responses during an oral glucose tolerance test (3.5 weeks, ANOVA). After 4 weeks a hyperinsulinaemic-euglycaemic clamp study was conducted. (d—f) Glucose infusion rate (GIR), glucose disposal rate (GDR) and insulin-stimulated glucose disposal rate (IS-GDR) were significantly enhanced. g, Hepatic glucose output (HGO) suppression (%). h, Plasma fatty acid concentration during the clamp was significantly lower in SRT1720-treated animals (ANOVA). B, basal conditions. C, clamp conditions. i, Glucose excursion during a PTT was reduced, indicating reduced gluconeogenic capacity. All studies consisted of n ≥ 5 rats per group. Statistics were conducted as student t-test unless noted otherwise; asterisk P < 0.05, double asterisk P < 0.01, and triple asterisk P < 0.001. Data are expressed as mean ± s.e.m.