新型抗代谢综合征药物的筛选及发现

发布时间：2018-07-12 08:04

本文选题：代谢综合征 + RCT　；参考：《北京协和医学院》2015年博士论文

【摘要】：代谢综合征是指人体内的蛋白质、脂肪、碳水化合物等物质发生代谢紊乱,从而造成的一组复杂的代谢紊乱症候群,是导致糖尿病、心脑血管疾病等的危险因素。其中心脑血管疾病是当今世界范围内发病率、致死率最高的重大疾病之一,动脉粥样硬化(Atherosclerosis, AS)是其主要的病理基础,而脂质代谢尤其是胆固醇代谢紊乱又是AS的主要致病因素。早期抗动脉粥样硬化药物的研究以调脂为主要特征,他汀类药物是典型代表。这类药物虽然可以有效地降低胆固醇,但是其总的有效率仅为30-40%。糖尿病(DM, diabetes mellitus)是代谢综合征中一个非常重要的危险因素,现已成为第三大危害人类健康的疾病。过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor y, PPARy)在调节糖脂代谢稳态上具有重要的作用,是目前临床使用的噻唑烷二酮(Thiazolidinediones, TZDs)类药物的分子靶点。然而,TZDs完全激活PPARγ可产生一系列不期望的副作用,如体重增加、水肿和组织损伤等。因此,开发兼能调节糖代谢和脂代谢的新型药物极具前景。近年来,脂代谢过程中的关键途径——胆固醇逆向转运(reverse cholesterol transport,RCT)通路及选择性PPARγ调节剂(Selective PPARγ Modulator, SPPARyM)受到了越来越多的关注。鉴于此,我们进行了靶向RCT的先导化合物的筛选、药理学、机制研究以及PPARγ选择性激动剂虚拟筛选模型的构建与应用研究：1.靶向RCT的先导化合物改善脂质和葡萄糖代谢的药理学及机制研究RCT是指胆固醇由外周组织转运回肝脏进行再循环或以胆汁酸的形式随粪便排出体外的过程。ABCA1、ABCG1是ATP结合盒转运体超家族成员,介导细胞内磷脂和游离胆固醇转运到贫脂或无脂的ApoA-I,从而促进HDL的生成,启动RCT过程。SRB1为功能性的高密度脂蛋白受体。这些蛋白都是调控RCT过程的关键因子。本论文的第一部分工作是以实验室前期构建的ABCA1/SRB1表达上调剂筛选模型为基础,对国家新药(微生物)筛选实验室的化合物库进行筛选,得到了活性化合物E17241和E22840,它们能够在体外分子水平调节参与胆固醇代谢、转运、分泌以及脂肪酸代谢的蛋白表达,并且能够有效地促进巨噬细胞中的胆固醇外排,减少巨噬细胞泡沫化的发生,减少细胞内脂质堆积。在动脉粥样硬化模型ApoE-/-、鼠体内,E17241和E22840均能够降低血浆中胆固醇、甘油三酯以及LDL的水平,增加HDL的含量,同时主动脉和心脏流出道中的动脉粥样硬化斑块也明显减少。另外,小鼠肝脏内的脂质含量也明显下降,通过对肝脏细胞中的相关蛋白表达的检测说明E17241和E22840是通过调节ABCA1、ABCG1、SRB1等蛋白来将胆固醇逆转运至肝脏中进行代谢。除此之外,我们还发现这两个活性化合物能够激活PPARy,其中E17241不仅能够显著降低ApoE-/-小鼠的血糖浓度,还能够降低2型糖尿病模型KKAy小鼠的空腹血糖及胰岛素水平,改善其葡萄糖耐受能力和胰岛素敏感性。不仅如此,在KKAy小鼠体内,E17241还能够缓解其由于肥胖造成的高血脂,减少肝脏中的胆固醇含量。相对于罗格列酮,它对肝、肾的损伤较小。另外,我们还发现该化合物之所以能够上调ABCA1和SRB1的表达,部分原因可能是其通过PKC/PKA通路激活了PPARs和LXRs等核受体,并且增加了SRB1蛋白的稳定性。化合物E17241和E22840的活性均为首次报道,可作为靶向RCT的先导化合物,在体内外发挥对胆固醇代谢和葡萄糖代谢的调节作用,具有进一步开发成为抗动脉粥样硬化药物的潜能,其中E17241还有望发展成为治疗糖尿病并同时缓解其伴随的高血脂和脂肪肝的新型候选药物。2.PPAR丫选择性激动剂的虚拟筛选及药理学研究PPARy是配体结合的核受体超家族中的一员,主要分布在白色和棕色脂肪,它在调节脂肪细胞分化、脂质代谢、维持葡萄糖体内平衡和胰岛素敏感性中发挥重要作用。完全激活PPARy可诱导多组织非特异性基因的表达,而SPPARγM则能够选择性地调控某些特定基因的转录。目前有许多具有PPARγ部分激动活性的化合物在临床前试验中表现出与完全激动剂相当的胰岛素增敏作用,但是具有较弱的或基本不产生TZDs的副作用。本论文的第二部分工作主要是寻找和发现新型的PPARy选择性激动剂。我们建立了基于药效团的虚拟筛选工作流程,对国家新药(微生物)筛选实验室的化合物库进行了40,000样次筛选,获得了活性化合物F12016和F12025。这两个化合物是对PPARγ亚型具有选择性,激动PPARγ的最大效能也只有罗格列酮的20-30%,并且能够拮抗罗格列酮的激动作用。此外,F12016和F12025能够促进肝细胞和脂肪细胞代谢葡萄糖,同时明显减少了对脂肪细胞分化的诱导。它们由于与PPARγ相互作用的方式与罗格列酮不同,因此会产生不同的构象改变,并募集特定的辅因子,从而不同程度的调节靶基因的表达。在白发性2型糖尿病模型KKAy小鼠体内,F12016能够明显降低小鼠的空腹血糖,改善葡萄糖耐受性和胰岛素敏感性,但不会引起明显的体重增加。与罗格列酮相比,F12016还能够明显减少对成骨细胞分化的抑制作用。我们还研究了F12016和F12025与PPARy的相互作用模式,并初步探讨了其既能保留药理学作用又能减少副作用的原因可能是由于其相当程度的抑制了CDK5介导的PPARγ Ser273磷酸化。因此,化合物F12016和F12025可作为PPARγ选择性激动剂,有可能发展成为新型抗糖尿病的先导化合物。综上,本论文获得了靶向RCT的先导化合物E17241和E22840,在分子、细胞水平确证了其生物学活性并进行了体内药理学研究；构建了适用于高通量筛选PPARγ选择性激活剂的虚拟筛选模型,通过筛选得到了活性较好的F12016和F12025,确证了其体内外生物学活性并初步探讨了其机制。本论文工作为寻找新型抗代谢综合征药物奠定了基础,具有重要的理论和实践意义。
[Abstract]:Metabolic syndrome is a complex metabolic disorder syndrome caused by metabolic disorders in the body, such as protein, fat, carbohydrate, etc., which is a risk factor for diabetes, cardiovascular and cerebrovascular diseases, among which cardiovascular and cerebrovascular diseases are one of the most fatal diseases in the world. Atherosclerosis (AS) is the main pathological basis, and lipid metabolism, especially cholesterol metabolism disorder, is the main pathogenic factor of AS. Early anti atherosclerotic drugs are mainly characterized by lipid regulation, statins are typical. Although these drugs can effectively reduce cholesterol, but their total The effective 30-40%. diabetes (DM, diabetes mellitus) is a very important risk factor in the metabolic syndrome. It has now become the third major health hazard. Peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor y, PPARy) is important in regulating the homeostasis of glycolipid metabolism. The role is the molecular target of Thiazolidinediones (Thiazolidinediones, TZDs), which is currently used clinically. However, the complete activation of PPAR gamma by TZDs can produce a series of undesirable side effects, such as weight gain, edema and tissue damage. Therefore, the development of new drugs which can regulate glucose metabolism and lipid metabolism is very promising. The key pathways in metabolic processes - reverse cholesterol transport (RCT) pathway and selective PPAR gamma modulator (Selective PPAR gamma Modulator, SPPARyM) have attracted more and more attention. In view of this, we have screened the precursor compounds for targeting RCT, pharmacology, mechanism and PPAR gamma selectivity. Study on the construction and application of a virtual screening model for agonists: 1. the pharmacological and Mechanism Research on the improvement of lipid and glucose metabolism by the pilot compound targeting RCT RCT refers to the transfer of cholesterol from peripheral tissue to the liver for recirculation or in the form of bile acid in the form of fecal excretion of.ABCA1, ABCG1 is a ATP binding cassette transporter. Members of the family, which mediate intracellular phospholipids and free cholesterol transport to poor fat or fat free ApoA-I, thus promote the generation of HDL and start the RCT process as a functional high-density lipoprotein receptor. These proteins are the key factors in the regulation of RCT processes. The first part of this paper is to increase the expression of ABCA1/SRB1 in the early laboratory. On the basis of the agent screening model, the compound library of the national new drug (microorganism) screening laboratory is screened and the active compounds E17241 and E22840 are obtained. They can regulate the protein table of cholesterol metabolism, transport, secretion and fatty acid metabolism at the molecular level in vitro, and can effectively promote the cholesterol in macrophages. In the atherosclerotic model ApoE-/-, E17241 and E22840 can reduce the levels of cholesterol, triglycerides and LDL in plasma, increase the content of HDL, and also decrease the atherosclerotic plaques in the aorta and the heart outflow tract. In addition, the lipid content in the liver of mice also decreased significantly. By detecting the expression of related proteins in the liver cells, E17241 and E22840 were metabolized by reversing cholesterol by regulating ABCA1, ABCG1, SRB1 and other proteins. In addition, we also found that these two active compounds can activate PPARy, of which E17241 is not. It can only significantly reduce the blood glucose concentration in ApoE-/- mice and reduce the fasting blood glucose and insulin level in type 2 diabetes model KKAy mice, improve their glucose tolerance and insulin sensitivity. Not only so, in KKAy mice, E17241 can also alleviate hyperlipidemia caused by fat fat and reduce the cholesterol content in the liver. Relative to rosiglitazone, it has less damage to the liver and kidney. In addition, we also found that the compound can increase the expression of ABCA1 and SRB1, partly due to the activation of PPARs and LXRs nuclear receptors through the PKC/PKA pathway, and the increase of the stability of SRB1 protein. The activity of compound E17241 and E22840 is reported for the first time. As a pilot compound for targeting RCT, it plays a role in regulating cholesterol metabolism and glucose metabolism in vivo and in vivo and has the potential to be further developed as an anti atherosclerotic drug, and E17241 is also expected to develop into a new candidate drug,.2.PPAR, for the treatment of diabetes and the simultaneous release of hyperlipidemia and fatty liver. The virtual screening and pharmacological study of selective agonists PPARy is a member of the ligand binding nuclear receptor superfamily, mainly distributed in white and brown fat. It plays a vital role in regulating adipocyte differentiation, lipid metabolism, maintaining glucose homeostasis and insulin sensitivity. Fully activating PPARy can induce multi tissue nonspecific. The expression of the heterosexual genes, while SPPAR gamma M can selectively regulate the transcription of certain specific genes. Many of the compounds with PPAR gamma partial excitant activity present in preclinical trials show the insulin sensitization equivalent to complete agonists, but there are weak or basic side effects that do not produce TZDs. Second Part of the work is to find and discover new PPARy selective agonists. We set up a virtual screening workflow based on the pharmacophore, and screened the compound library of the national new drug (microorganism) screening laboratory for 40000 times. The two compounds, active compounds and F12025., were selected for the selection of PPAR gamma subtypes. The maximum efficiency of PPAR gamma is only the 20-30% of rosiglitazone, and it can antagonize the agitation of rosiglitazone. In addition, F12016 and F12025 can promote the metabolism of glucose in liver and fat cells, and obviously reduce the induction of adipocyte differentiation. They are different from rosiglitazone in the way of interaction with PPAR gamma. Therefore, different conformation changes will be produced and a specific cofactor is raised to regulate the expression of the target gene in varying degrees. In the white hair type 2 diabetes model KKAy mice, F12016 can significantly reduce the fasting blood glucose in mice, improve glucose tolerance and insulin sensitivity, but do not cause significant weight gain. F12016 can also significantly reduce the inhibitory effect on osteoblast differentiation. We also studied the interaction patterns of F12016 and F12025 and PPARy, and preliminarily discussed the reason that it could retain the pharmacological action and reduce the side effects because of its considerable inhibition of the CDK5 mediated phosphorylation of PPAR gamma Ser273. Therefore, compounds F12016 and F12025 can be used as PPAR gamma selective agonists and may develop into a new type of antidiabetic precursor compound. In this paper, E17241 and E22840, a precursor of target RCT, were obtained in this paper, and their biological activities were confirmed at the molecular and cell levels. The virtual screening model of PPAR gamma selective activator was screened and the active F12016 and F12025 were obtained by screening. The biological activity of the active agent was confirmed and its mechanism was preliminarily discussed. This work has laid the foundation for finding new anti metabolic syndrome drugs, which has important theoretical and practical significance.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R589

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