Discovery And Pharmacology Mechanism Investigation Of Anti-T2DM Lead Compounds By Targeting Pancreatic ?-cell Function Promotion And Hepatic Gluconeogenesis Inhibition

Diabetes mellitus(DM)is a chronic,systemic,metabolic disease mainly characterized by hyperglycemia with insulin resistance and deficiency.DM is caused by long-term damages of glucose,lipid and protein metabolisms induced by genetic and environmental factors.DM mainly consists of insulin-dependent type 1 diabetes mellitus(T1DM),insulin-independent type 2 diabetes mellitus(T2DM)and gestational diabetes mellitus(GDM).Among them,the incidence of T2 DM accounts for more than 90% of the total cases,and the number of patients is increasing remarkably in the past few decades.This disease has become one of the most health-threating diseases.Although a series of anti-T2 DM drugs are being currently used,both oral and injectable dugs exist different side effects,thus triggering the urgent need for safe agents in the treatment of T2 DM.Therefore,study on the anti-T2 DM drugs with high efficiency and low side-effects is of potently scientific significance and practical value.Insulin resistance is implicated with the entire course of T2 DM,and the damage of ?-cell function is more likely to increase the progression of the disease.Glucose-lipid toxicity,mitochondrial dysfunction,stress and inflammatory reaction can induce the apoptosis of pancreatic ? cells,accelerate ?-cell death and reduce the ?-cell number,finally attenuating insulin secretion.Thus,it is of great significance to enhance the protection of ? cells and improve the function of ? cells in the treatment of T2 DM.Hepatic gluconeogenesis is an important source of endogenous glucose in the body,whose physiological significance is to maintain body's glucose homeostasis avoiding the occurrence of hypoglycemia during fasting.However,the insulin resistance,insulin secretion deficiency and hyperglucagonemia in T2 DM lead to excessive activation of gluconeogenesis and glucose output.Since the glucose generated from gluconeogenesis is the most important source of high blood glucose in T2 DM patients,inhibition of hepatic gluconeogenesis and endogenous glucose production is thus expected to be an optimal strategy in the treatment of T2 DM.Given the potencies of ?-cell defect and gluconeogenesis abnormal activation in hyperglycemia generation,we focused on the discovery of new anti-T2 DM lead compounds by targeting ?-cell function promotion and hepatic gluconeogenesis inhibition,while the further study of the lead compounds on pharmacodynamic evaluation and mechanism investigation are expeted to be helpful to explore the pathogenesis of T2 DM and lay the foundation to find more effective and safe anti-T2 DM drugs.As for the strategy of ?-cell function promotion,we focused on the study on voltage-gated potassium channel Kv2.1.Kv2.1 plays a pivotal role in stimulating insulin secretion and ?-cell survival.Our laboratory previously established high-throughput screening platform for Kv2.1 inhibitor,and small molecule SP6616(Ethyl-5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methy l-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2–a]pyrimidine-6-carboxylate)was determined as a novel Kv2.1 inhibitor.In the current work,we found that SP6616 exhibited activities in promoting both insulin secretion activation and ?-cell apoptosis resistance in INS-1 cell lines,and effectively improved glucose homeostasis in db/db mice by increasing insulin secretion and preventing ?-cell apoptosis in vivo.Pharmacological mechanism study revealed that SP6616 inhibited Kv2.1 leading to the open of voltage-gated calcium channels and the increase of extracellular calcium influx into cells,and activating the glucose-stimulated insulin secretion,which is in accordance with the report that potassium channel inhibition increases insulin secretion through regulation of calcium.Moreover,we also investigated the regulation of Kv2.1 against ?-cell apoptosis with SP6616 as a molecular probe.For the first time,we discovered that the regulation of Kv2.1 against ?-cell apoptosis was related to the protein kinases C(PKC)/extracellular-regulated protein kinases 1/2(ERK1/2)and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)pathways.SP6616 was capable of promoting both insulin secretion activation and ?-cell apoptosis resistance in vitro and in vivo.With potent anti-T2 DM biological activity and clear mechanism,it is expeted that SP6616 exhibited the potential as a new drug lead compound in anti-T2 DM drug development.As the strategy of hepatic gluconeogenesis inhibition is concerned,weconstructed a screening platform using the mouse primary hepatocytes.By screening out over 7,000 compounds from the in-house chemical compound library,we determined that two active compounds DMT(2-(2,4-Dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3',2': 4,5]thieno[3,2-d]pyrimidin-4(1H)-one)and QVO((E(-3-)2-(quinolin-4-yl)vinyl)-1H-indol-6-ol)could antagonized the elevation of the glucagon-stimulated gluconeogenesis.In the study,DMT could alleviate the pathological events of db/db mice diabetic,in that DMT reduced fasting blood glucose and hemoglobin A1c(HbA1c)levels,improved oral glucose tolerance test(OGTT)and pyruvate tolerance test(PTT),and transcriptionally inhibited glucose-6-phosphatase(G6Pase)and phosphoenolpyruvate carboxykinase(PEPCK)genes in liver.Furthermore,we determined that DMT activated G?q/phospholipase C(PLC)/inositol 1,4,5-trisphosphate receptor(IP3R)signaling pathway to increase the release of Ca2+ from endoplasmic reticulum into cytoplasm,leading to the regulation of PI3K/AKT/forkhead box protein O1(FOXO1)signaling and inhibition of gluconeogenesis.It is expected that DMT may become a potential new anti-T2 DM lead compound.Structurally,QVO exhibited the chemical structure skeleton similar to that of berberine,and indirectly stimulated the activity of AMP-activated protein kinase(AMPK)and inhibited gluconeogenesis.However,the mechanism of QVO action was quite different from that of berberine,in that QVO regulated the upstream of AMPK,like liver kinase B1(LKB1),calcium/calmodulin dependent protein kinase kinase ?(CaMKK?)and mitochondrial respiratory pathway.Due to the complex chemical synthesis route and low yield of QVO,we designed the prodrug IVQ(4-[2-(1H-indol-3-yl)vinyl]quinolin),which has been confirmed by the pharmacokinetic assay,in that IVQ was effectively converted into QVO in mice and rats.In the current work,we investigated the activity of the prodrug IVQ hydrochloride in both db/db and ob/ob mice.IVQ hydrochloride reduced fasting blood glucose and HbA1 c levels and improved OGTT and PTT.Thus,IVQ is expected to be a precursor of anti-T2 DM drug lead compound for further development.In summary,the thesis included the discovery,pharmacodynamic evaluation and mechanism investigation of active compounds SP6616,DMT and QVO against T2 DM.We have found three drug lead compounds by targeting ?-cell function promotion and hepatic gluconeogenesis inhibition,our results have provided new valuable structural information and research ideas for novel anti-T2 DM drug discovery.