新型E3泛素连接酶WSB-1抑制剂的设计、合成和抗肿瘤转移活性研究

Tumor metastasis is considered to be closely related to hypoxiic microenvironment, which has been confirmed to promote the tumor metastasis via directly enhancement of the potential motion ability of tumor cells themselves. Therefore, by targeting to hypoxic microenvironment, we have identified the E3 ubiquitin ligase WSB-1 as a key target protein for tumor metastasis, and successfully discovered a lead compounds Dxw-W15 as WSB-1 inhibitor explicitly with antitumor metastasis activities. Based on the basic skeleton of Dxw-W15, in this project, we will further perform the rational drug design and structural optimization of Dxw-W15 by the combined approaches of bioisosterism, skeleton hopping, drug-likeness analysis, molecular docking. After organic synthesis and biological evaluation of the terget compounds, we expect to obtain the highly active WSB-1 inhibitors with potent antitumor metastasis activities, which can be used as molecular probes to clarify the role of HIF-1-WSB-1 pathway in titumor metastasis and its exactly mechanism of WSB-1 inhibition. Meanwhile, the interaction mode between WSB-1 and the highly active WSB-1 inhibitors will be demonstrated by the NMR technology, to provide direct evidence that specific binding between each other. Therefore, the implementation of this project and the innovative found will provide the experimental basis to confirm the WSB-1 as novel an antitumor metastasis target under hypoxia. Also, this work can be of great reference significance for the discovery of other novel tumor metastasis regulatory proteins targeting hypoxia microenvironment, as well as their specific small molecular inhibitors.

肿瘤转移被认为与缺氧微环境关系密切，已证实缺氧可增强肿瘤细胞本身运动潜而促进转移。前期本课题针对缺氧微环境的研究，发现了全新抗肿瘤转移的靶蛋白WSB-1，并以此为切入点，通过基于WSB-1结构的虚拟筛选、活性测试获得了具有明确抗肿瘤转移活性的新型WSB-1抑制剂Dxw-W15。本项目拟进一步以其为基本骨架，综合运用电子等排、骨架迁跃、类药性分析、分子对接等技术对其进行合理设计，在有机合成、生物活性评价基础上，以期获得WSB-1抑制活性高、体内外抗肿瘤转移活性强的目标分子。并以其为分子探针，进一步阐明靶向WSB-1的抗肿瘤转移活性的独特机制。同时，通过NMR技术研究WSB-1蛋白与配体的结合模式，为两者的特异性结合提供直接证据。本课题的顺利实施将为证实WSB-1成为抗肿瘤转移的新靶标提供实验依据，同时对发现其他新型肿瘤转移调控蛋白及其小分子抑制剂的研究工作提供了借鉴。

本研究围绕肿瘤转移与缺氧环境的关系，受低氧调控的WSB1蛋白可泛素化降解RhoGDI2，从而促进肿瘤转移。本研究采用表型筛选的方法，成功设计、合成了64个化合物，并发现了具有明显抗肿瘤转移活性的分子。其中，包括五元环内酯化合物1a（对KHOS和H460细胞迁移率分别为0.53和0.17）；以及以2-氨基吡啶为骨架，进行结构合理优化的获得的化合物4、B1、B4-B6等分子（对KHOS和H460细胞迁移率达到了0.17）。随后优选抗肿瘤转移活性优异的分子开展作用机制研究，发现化合物4、B12等分子对WSB1下游RhoGDI2的泛素化降解具有明显的抑制作用，且对WSB1过表达的KHOS和H1299细胞具有明显的迁移、侵袭抑制活性。其中化合物4表现了较好的PK代谢性质，在原位接种乳腺癌 4T1 细胞的小鼠体内肺转模型上，其对肿瘤转移灶点的抑制率为63.2%。为了进一步证明该类分子与WSB1蛋白的直接结合作用，开展了蛋白纯化工作，在尝试了不同条件后，发现纯化后的蛋白不稳定，无法获得高纯度以及高浓度的WSB1蛋白开展体外与小分子的相互作用验证实验。随后采用计算机建模的方式，构建了WSB-1与底物蛋白识别区域（WD40重复结构域），探索了化合物4与WSB1蛋白的可能作用位点。综上，本研究发现了一类新型抗肿瘤转移的WSB1-RhoGDI2调节剂分子，证明了通过调控该通路抗肿瘤转移的可行性，为抗低氧驱动的肿瘤转移提供了新的研究基础和思路。