Discovery And Molecular Pharmacology Of New Drug Leaders For Regulating Glucose And Lipid Metabolism

Metabolic syndrome is a complex metabolic disorder which caused by metabolic disorder of glucose and fat.Metabolic syndrome is a key risk factor for diabetes and cardiovascular disease.Diabetes and cardiovascular disease are the major cause of mortality and disability worldwide.The representative drug for diabetes,Thiazolidinediones,which could regulate glucose metabolism by activating Peroxisome Proliferator Activated Receptor ?,but may cause weight gain,lipid accumulation and tissue damage.And the most famous drug for cardiovascular disease,statins,even effectively lower the cholesterol level in the serum,however,they showed a series of side effects,such as liver injury,muscle injury and hyperglycemia.Therefore,it's very promising to develop new drugs that can improve glucose and lipid metabolism with less side effects.Sirtuin 1(SIRT1)is an NAD+-dependent protein deacetylase that plays a critical role in controlling energy metabolism,stress response and aging.Kruppel-like factor 2(KLF2)is an important zinc-finger transcription factor that maintains endothelial homeostasis by its anti-inflammatory,-thrombotic,-oxidative,and-proliferative effects in endothelial cells.In light of the potent vasoprotective effects of SIRT1 and KLF2,modulating SIRT1 and KLF2 activity or expression could give rise to new therapeutic strategies to treat diabetes and cardiovascular diseases.However,pharmacological and molecular regulators for SIRT1 or KLF2 are scarce.In view of this,we established high-throughput screening models targeting SIRT1 and KLF2 to identify novel activators.Our work is described as below.1.SIRT1 activator protects against glucose and lipid metabolism disordersSIRT1,an NAD+-dependent protein deacetylase,mediates the effects of caloric restriction on lifespan and metabolic pathways in various organisms.Recent studies demonstrated that enhancing SIRT1 expression or activity may modulate glucose and lipid metabolism.However,pharmacological and molecular regulators for SIRT1 are scarce.The aim of research work in the first part is to find novel small molecular modulators of SIRTI to regulate glucose and lipid metabolism.High throughput screening assay was established to find SIRT1 activators.E6155,a piperazine 1,4-diamide compound,was first identified as a new small molecular activator of SIRTI.We further found that E6155 significantly upregulated glucose uptake in cultured mouse liver cells and skeletal muscle cells through increasing SIRT1 deacetylase activity.In type 2 diabetic KKAy mice,E6155 treatment markedly decreased the level of fasting glucose.Moreover,E6155 improved oral glucose tolerance and insulin tolerance.Euglycemic clamp and the homeostasis model assessment of insulin resistance index showed that E6155 ameliorated the insulin resistance and increased insulin sensitivity in diabetic mice.Mechanistically,we observed that the antidiabetic effects of E6155 were involved in SIRT1 dependent activation of LKB1/AMPK and IRS1/AKT pathways.Our findings identified E6155 as a novel SIRT1 activator and suggested that E6155 could be a promising drug candidate for treating insulin resistance and diabetes.In addition,using the optimized high throughput screening,we identified E1231,a piperazine 1,4-diamide compound,as a SIRT1 activator through screening 12 000 compounds with an EC50 value of 0.83 ?M.Surface plasmon resonance assay showed E1231 could interact with recombinant human SIRT1 protein.Immunoprecipitation assay demonstrated that E1231 could deacetylate LXR?.Western blot assay revealed that E1231 increased ABCA1 expression in RAW 264.7 cells dependent on SIRT1 and LXRa.Fluorescence labeled cholesterol assay demonstrated that E1231 promoted cholesterol efflux and inhibited lipids accumulation in RAW 264.7 cells through SIRT1 and ABCA1.In golden hamster hyperlipidemia model,E1231 treatment decreased TC and TG levels in both serum and liver,and increased cholesterol content in feces.Meanwhile,E1231 increased ABCA1 and SIRT1 protein expression in the liver of hamsters.In ApoE-/-mice,E1231 treatment reduced atherosclerotic plaque development compared with model group.Our findings identified a novel SIRT1 activator E1231 and elucidated its beneficial effects on lipid and cholesterol metabolism.2.Pharmacological kruppel-like factor 2 activator that represses vascularinflammation and atherosclerosisThe transcription factor Kruppel-like factor 2(KLF2)is a critical anti-inflammatory and anti-atherogenic molecule in vascular endothelium.Enhancing KLF2 expression and activity improves endothelial function and prevents atherosclerosis.However,the pharmacological and molecular regulators for KLF2 are scarce.In the second part of the thesis,high-throughput drug screening based on KLF2 promoter luciferase reporter assay was performed to screen KLF2 activators.By screening 2 400 compounds in the Spectrum library,we identified suberanilohydroxamic acid(SAHA),also known as vorinostat as a pharmacological KLF2 activator through myocyte enhancer factor 2.We found that SAHA exhibited anti-inflammatory effects and attenuated monocyte adhesion to endothelial cells inflamed with tumor necrosis factor alpha.We further showed that the inhibitory effect of SAHA on endothelial inflammation and ensuing monocyte adhesion was KLF2 dependent using KLF2-deficient mouse lung endothelial cells or KLF2 small interfering RNA-depleted human endothelial cells.Identified KLF2 activator,SAHA,was orally administered to ApoE-/-mice to evaluate anti-atherosclerotic efficacy.Importantly,we observed that oral administration of SAHA reduced diet-induced atherosclerotic lesion development in ApoE-/-mice without significant effect on serum lipid levels.These results demonstrate that SAHA has KLF2-dependent anti-inflammatory effects in endothelial cells and suggests that the anti-cancer drug SAHA could be repurposed as an effective therapeutic agent for atherosclerosis.Using high-throughput luciferase reporter assay to screen for KLF2 activators,we also get another positive hit,tannic acid(TA),a polyphenolic compound,as a potent KLF2 activator that attenuates endothelial inflammation.Mechanistic studies suggested that TA induced KLF2 expression in part through the ERK5/MEF2 pathway.Functionally,TA markedly decreased monocyte adhesion to ECs by reducing expression of adhesion molecule VCAM1.Using lung ECs isolated from KLF2+/-and KLF2+/-mice,we showed that the anti-inflammatory effect of TA is dependent on KLF2.Collectively,our results demonstrate that TA is a potent KLF2 activator and TA attenuated endothelial inflammation through upregulation of KLF2.Our findings provide a novel mechanism for the well-established beneficial cardiovascular effects of TA and provide the proof of concept that KLF2 activation could be a promising therapeutic strategy for treating atherosclerosis.In conclusion,we used HTS model of SIRT1 and KLF2 to identify drug candidates for treating metabolic syndrome.The results elucidated the beneficial effects of SIRT1 and KLF2 activators on glucose and lipid metabolism and suggested that they might be developed as a novel drug for treating diabetes and atherosclerosis.