糖原合成酶激酶-3β（GSK-3β）是糖尿病病程中的关键调控激酶，其抑制剂可用于治疗Ⅱ型糖尿病，提高激酶选择性是成药的关键。非ATP竞争抑制是解决这一问题的新思路和新途径。申请人发现的苯并噻氮杂卓化合物是具有较好活性和选择性的新型GSK-3β非ATP竞争抑制剂，已在EJMC和BMCL发表论文2篇,专利授权1项。但对靶酶的作用位点和分子机制尚不清楚，为基于受体的合理药物设计带来困难。本课题拟以其为先导物，综合运用基于靶点结构/配体的多种CADD技术，设计合成多样性新结构分子。结合分子和细胞水平的生物评价，研究构效关系和降糖活性。同时综合运用虚拟突变和点突变技术探索目标分子可能的变构作用位点；通过结晶学研究获取共晶复合物，以揭示小分子与靶酶的分子作用机制。本项目的实施将可能获得源头创新的系列高选择性活性化合物，并阐明明确的分子作用机制，为最终开发原创新药提供物质基础和理论指导。

Glycogen synthase kinase-3β （GSK-3β）is widely recognized as a relevant player in the pathogenesis of diabetes. Therefore, this enzyme constitutes a highly attractive therapeutic target for the development of inhibitors as new promising drugs for the treatment of this disease. To which non-ATP competitive inhibitors represent an effectively therapeutical approach due to their good specificity. Fortunately, the proposer has successfully found a series of novel small molecule benzothiazepinones (BTZ) as non-ATP competitive inhibitors of GSK-3β, which has been confirmed having good activities and selectivities. Three China patents have been applied for and one already authorized, as well as two papers had been published in EJMC and BMCL, respectively. However, the real binding site and mechnism of BTZ compounds remain unknown. So this study will explore the allosteric modulation cavities with their identification and characterization to find the binding sites of BTZs and their mode of action. The research plan includes design and synthesis of large amount of new active molecules with diversity of chemical structures based on comprehensive utilization of computer aided drug design technology as pharmacophore searching, scaffold hopping, structure-based and ligand-base optimization of BTZ compounds. Moreover the structure-activity relationships (SAR) based on the biological activities will be investigated, as well as the hypoglycemic activity and mechanism of action evaluated. On the other side, the possible allosteric site of BTZ will be probed by using the techniques of protein virtual mutagenesis and point mutation, and the X-ray crystallographic data from the analysis of the co-crystallization complex of BTZ and GSK-3β would prove a clear map of action mechanism. As the results, the obtaind potential candidates and the clear molecular mechanism of action will provide the original material foundation and theoretical guidance for the development of new drugs.

本工作按计划完成了相关研究内容，达到了预期目标，并拓展完成了申请书中没有的抗肿瘤、抗炎、神经保护方面的药效及分子机制等研究内容。运用CADD技术完成了先导结构的优化和新结构类型的分子设计，共合成近400个结构多样性目标化合物，通过体外酶活评估和激酶谱筛选已获得一批具有高活性、高选择性的新型GSK-3β非ATP竞争型化合物，拓展了活性结构类型，丰富了活性分子的结构多样性；结合酶动力学分析确证了活性分子的底物竞争、变构抑制、共价结合等不同作用模式；通过Docking技术揭示了其与靶标蛋白的分子结合机制，完善了构效关系，明确了药效基团、取代基、立体构型等对活性的影响；酶动力学分析进一步明确了非ATP竞争抑制剂结构的微小变化都可能异致作用模式的改变，提示靶酶的底物作用区、变构抑制位点和共价结合区可能相距较近。进一步通过细胞模型和动物模型的药效评价，确证了一批明显具有不同药效作用、且类型各异的新结构、新机制的高活性候选分子，这些药效作用包括降糖、抗肿瘤、抗炎和神经保护功能；进一步运用分子生物学和细胞生物学实验方法研究了信号通路及分子作用机制。拓展的多种结构均为首次发现具有GSK-3β抑制活性的新结构类型，或首次报道具有体内外抗卵巢癌增殖活性的非ATP竞争型抑制剂。这些研究结果为进一步开发针对特定疾病的创新药物提供了新的方向和基础。进行了初步的结晶学研究。建立了GSK-3β蛋白酶的表达和纯化体系，开展了活性分子与靶蛋白共晶复合物的结晶学研究，成功培养出了靶蛋白晶体，优化了复合物共晶条件，但目前尚未获得与小分子的共晶复合物。以通讯作者发表3篇SCI论文（EJMC，BMCL，Tetrahedron Letters），另有两篇正在投稿中。申请中国发明专利1项，协助培养博士研究生2名。