针对cADPR-RyR-钙离子信号通路的小分子设计、合成及其作为潜在糖尿病治疗药物的研究

Insulin secretion deficiency is one of the most important causes of type 2 diabetes mellitus (T2DM). Promoting insulin secretion could decrease patients’ high blood glucose and ameliorate diabetes-related complications. The widely used insulin secretagogues can promote insulin secretion mainly through inducing the influx of extracellular calcium and then raising the intracellular calcium level. RyR is a calcium channel on the endoplasmic reticulum membrane, and can be taken as a possible target for insulin secretagogues because it was recently reported to induce insulin secretion by mediating calcium release from intracellular stores. However, cADPR, an endogenous modulator of RyR, cannot be used as a molecular probe for mechanistic study owing to its poor membrane permeability and stability.. The applicant has identified GAPDH as a new regulating protein for the cADPR-RyR-calcium signaling pathway, which can be used as a target for novel secretagogue development. This project aims to design, synthesize heterocyclic cADPR specific agonists and antagonists, and optimize the structures based on their pharmacological properties in protein and cells. Lead compounds with better membrane permeability and stability would be discovered and used to explore the structure-activity relationship. Afterwards, effects of lead compounds on insulin secretion and blood glucose level will be tested in cells and model animals respectively. Based on the druggability study of lead compounds, potential anti-diabetic drug candidates with novel mechanism would be obtained.

胰岛素分泌不足是2型糖尿病的发病机制之一，促进胰岛素分泌能够改善患者的高血糖状况，减少糖尿病并发症。临床常用的促泌剂主要是促使细胞外钙内流，提高胞内钙离子浓度，从而促进胰岛素分泌。RyR是细胞内质网膜上的一种钙通道，最新文献报道RyR介导的细胞内钙库释放同样可以促进胰岛素的分泌，是新型胰岛素促泌剂的靶标。然而，RyR的内源性调控分子cADPR膜透性和稳定性均较差，无法作为探针分子用于深入研究RyR对胰岛素分泌的影响。基于申请人所确认的cADPR-RyR-钙信号通路的全新调控蛋白GAPDH，我们将设计并合成特异性的杂环类cADPR激动剂或拮抗剂，根据蛋白和细胞水平的活性测试结果进行结构优化，得到膜透性和稳定性都有所提高的先导化合物，并总结其构效关系。在此基础上研究先导化合物在细胞水平对胰岛素分泌的影响，评估其对于模型动物的血糖调节作用，并初步研究其成药性，得到全新机制的潜在抗糖尿病药物。

环腺苷二磷酸核糖（cADPR）是一种普遍存在于动植物细胞内的第二信使分子，通过调控细胞内的钙离子信号通路来参与一系列重要的生理过程，如胰岛素分泌和动物社会行为的调控等等。目前，已有许多研究人员设计并合成了一系列cADPR结构类似物作为探针分子，用于研究cADPR调节钙信号通路的作用机制及下游生物学功能；然而已报道的cADPR-钙信号通路的探针分子均为核苷酸类结构，其合成和纯化较为复杂，且膜透性和稳定性一般较差。基于前期所确认的cADPR结合蛋白GAPDH，我们采用分子对接的方法进行虚拟筛选，获得了几个杂环类小分子作为苗头化合物；蛋白水平的SPR实验表明苗头化合物能够与靶标蛋白GAPDH产生特异性结合，而细胞实验表明其能够在完整细胞中产生与cADPR类似的钙激动活性。随后基于对化合物与蛋白质结合模式的分析，我们对苗头化合物进行了系统的结构优化，获得了活性显著提高的先导化合物（其钙激动活性与公认的cADPR激动剂cIDPRE基本相当），并系统地研究了其作用机制。本项目首次得到了杂环类cADPR-钙信号通路激动剂，使cADPR激动剂的结构类型更为多样化，有望为cADPR-钙信号通路相关的生理活性和作用机制研究提供全新的、具有更为优越的理化性质的探针分子。

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