基于Neddylation通路筛选治疗结直肠癌的新型候选药物靶点

Neddylation pathway is a newly-characterized posttranslational modification pathway that adds the ubiquitin-like molecule neural precursor cell expressed, developmentally downregulated 8 (Nedd8) to substrate proteins. Nedd8-activating enzyme (NAE) is an essential component of the neddylation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. MLN4924 is a small molecule inhibitor of NAE that disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by new mechanisms of action, such as the deregulation of S-phaseDNAsynthesis. Currently, MLN4924 is under consideration in multiple phase I studies to treat solid tumors, metastatic melanoma and various hematologic malignancies. However, some of the primary cancer cell lines with the low expression of NAE1 were highly resistant to MLN4924 treatment, indicating the presence of primary resistance to MLN4924 in cancer, and highlighting the need for precise patient selection according to the activation status of neddylation pathway. Similarly, treatmentinduced mutations of NAEβ/UBA3 were reported to lead to secondary resistance to MLN4924 in leukemia and colorectal cancer, further mandating the development of combinational regiments including novel neddylation pathway inhibitors. Our previous study demonstrated that some of the enzymes in neddylation pathway was upregulated/overactivated in colorectal cancer and probably served as therapeutic targets. In this project, we will reveal the clinical significance and independent prognostic value of the enzymes in neddylation pathway and conduct preclinical evaluation of inhibiting this pathway by targeting the enzymes using shRNA incolorectal cancer models of in vitro and in vivo. We will discover new small molecule inhibitors of the potential therapeutic targets and build 3D interaction models between these inhibitors and targets, through virtual screening the compounds in databases by using virtual screening tools. This project will focus on the relevance of neddylation pathway to colorectal cancer and discovery of new neddylation inhibitors, such as inhibitors of neddylation E2 or E3 and next-generation E1 inhibitors may provide additional choice for targeting therapy of colorectal cancer and overcoming the primary/secondary resistance to MLN4924.

针对泛素化和Neddylation通路设计抑制剂，是研发靶向药物治疗癌症的新方法。Nedd8激活酶NAE的抑制剂MLN4924已进入多种肿瘤的I期临床试验，具有广阔的应用前景。但是使用MLN4924治疗，部分结直肠癌和白血病细胞会发生原发性和诱导性抵抗。根据Neddylation通路信号传递特点，在NEA之外，此通路很可能存在更多的治疗靶点。我们近期发现，Neddylation通路在结直肠癌中过度激活，导致Nedd8修饰水平显著提高，促进肿瘤发展。本项目将在前期基础上深入研究，利用泛素化和Neddylation修饰、组织芯片、原位移植瘤模型、虚拟筛选、共结晶等技术，评价Neddylation通路已知成员与结直肠癌临床病理特征和预后的关联程度，基于此通路发掘新的候选药物靶点，筛选先导化合物。本研究将为克服MLN4924抵抗提供更多的靶点选择，为结直肠癌的诊治和新型靶向药物的研发提供科学基础。

针对泛素化和Neddylation通路设计抑制剂，是研发靶向药物治疗癌症的新方法。根据Neddylation通路信号传递特点，此通路很可能存在多个治疗靶点。本研究收集600例患者肿瘤和癌旁组织样本，制成组织芯片，利用免疫组化技术分析Neddylation通路的关键成员在结直肠肿瘤与正常组织的差异性。结合患者治疗和预后资料，分析这些分子表达情况与临床病理参数的相关性，收集预后随访资料，分析蛋白表达水平与预后生存时间的关系。选定较为关键、具备合适疏水口袋的关键分子作为候选靶点。虚拟筛选小分子化合物抑制剂，分析了化合物与大分子之间的作用模式，购买、合成化合物。利用体内体外泛素化、类泛素化等多种技术进行验证化合物对Neddylation通路的抑制效应，优选效应较为显著的先导化合物；鉴定先导化合物作用于结直肠癌细胞后关键蛋白质成分及其磷酸化、类泛素化等修饰的影响，分析其调控的通路；鉴定先导化合物对肿瘤细胞周期、增殖、凋亡等生物学行为的作用。利用稳定同位素标记氨基酸技术（SILAC）和蛋白质质谱技术初步鉴定了3个先导化合物处理之后的结直肠癌细胞的蛋白质情况。系统地揭示了Neddylation通路成员在结直肠癌中的状态及与结直肠癌临床病理参数的关联性，确认Neddylation通路主要成员对预后的判断价值，鉴别了结直肠癌发展所需的关键催化酶；基于新的潜在药物靶点的结构，虚拟筛选获得200多个小分子化合物抑制剂，确定了抑制剂的抗肿瘤作用；在蛋白质组学水平揭示候选药物靶点参与结直肠癌发展的分子机制；揭示了先导化合物处理后结直肠癌细胞的蛋白质表达情况。利用对接软件分析了先导化合物、药物靶点所形成复合物的可能结合模式和三维结构。总之本研究为结直肠癌的诊治和新型靶向药物的研发提供了新的科学基础。