双位点结合型CDC25B-CDK2/CyclinA相互作用调控分子的发现及作用机制研究

细胞分裂周期蛋白CDC25磷酸酶可以去磷酸化CDK/Cyclin复合物从而使其活化，进而调控细胞周期及细胞增殖，是一个极有前途的癌症治疗靶标。本课题为发现全新作用机制的CDC25抑制剂及亚型选择性探针分子，以CDC25B-CDK2/Cyclin A相互作用作为药物设计新靶标，基于CDC25B“泳池”及“热点区域”位点的结构和已有配体的药效团，分别确定各位点优势片段，并实施化学合成，运用CuAAC点击化学高效合成筛选技术，构建含有三唑Linker的双位点结合型片段组合库，经原位抑酶活性及CDC25B-CDK2/Cyclin A相互作用抑制活性（SPR法及AlphaLISA法）筛选发现活性分子；进而分别从分子水平、细胞水平及动物水平对其抑制剂活性及抗肿瘤活性进行评价，并进一步对活性化合物的作用机制及成药性进行探讨，为开发新型CDC25亚型选择性探针分子及精准靶向的抗肿瘤侯选药物奠定基础。

Cell division cycle (CDC) 25 phosphatases are promising targets for anticancer drug discovery as they are key phosphatases regulating cell cycle transition and proliferation via the interactions with CDK/Cyclin complexes. To date, all efforts to inhibit CDC25 phosphatases were focused on targeting the catalytic sites of these enzymes, which are unusually small and shallow without well-defined binding pockets, making CDC25s somewhat recalcitrant to drug discovery efforts. Discovery of bidentate CDC25B-CDK2/Cyclin A interaction modulators provided a more attractive strategy to design novel agents with therapeutic applications, by binding simultaneously in CDC25B “swimming pool” and “remote hotspot region”. Based on these findings, in this project, we propose CuAAC click-chemistry-based combinatorial libraries approach to discover bidentate CDC25B-CDK2/Cyclin A interaction modulators. Based on multiple-filter-based virtual screening, the fragments targeting “swimming pool” and “remote hotspot region” will be identified from a large compound collection respectively, which having a tolerant region (site) to allow attachment of an appropriate linker. The top-ranking binders and favorable linkers will be selected and be dissected into a set of alkyne-/azide-containing fragments. Initially, a library of 240-membered 1,4-triazoles between 10 alkynes and 24 azides is rapidly constructed using CuAAC reaction and tested against CDC25B and CDC25B-CDK2/Cyclin A interaction (in situ screening). From the preliminary inhibition data, the most potent library members will be individually resynthesized in large-scale for complete characterization and further assay of their inhibition activity against CDC25 phosphatases as well as anticancer activities both in vitro and in vivo. Investigations will also be conducted to estimate their ability to induce cell cycle arrest as well as apoptosis. Assays of ROS generation, mitochondrial dysfunction and DNA damage may help to explain the mechanism of action. Moreover, preliminary evaluation of druggability properties will reveal their potential as anticancer drug candidates. The binding modes of selected bioactive compounds will be investigated by molecular simulations. Further modifications and a new round of library design (using other bioorthogonal click reactions) will be performed to obtain second-generation compounds with improved properties. Our approach thus lays the foundation for discovery of “bidentate”-typed modulators with improved CDC25 subtype-selectivity, antitumour activities and favorable druggability.