RIP3作为抗肿瘤分子靶点及其抑制剂的发现与机制研究

Identification and validation of anticancer targets and discovery of new anticancer mechanisms and new chemical entities are very important approaches to R & D of innovative anticancer drugs. Receptor-interacting protein 3 (RIP3) is a serine/threonine protein kinase, which has aroused extensive research interests due to the finding that it acts as a molecular switch between apoptosis and necrosis (also called as necroptosis). However, it is unclear whether RIP3 is involved in tumor progression, and especially, whether it is suitable for an anticancer target. Moreover, as yet, no small-molecule inhibitors of RIP3 have been reported. Just recently, we have demonstrated, for the first time, the differential sensitivity of the RIP3-deficient A cells and the RIP3-proficient N cells to anticancer agents chimmitecan and SN38, suggesting a potential new role of RIP3 in topoisomerase I inhibitor-induced DNA damage/repair and cellular resistance to topoisomerase I inhibitors, probably independently of its regulation in the choice of cell death modes. We have also found that RIP3 is expressed at high levels in cancer cells originated from different human tissues. We have constructed the constructs of full-length RIP3 (wild-type), wild-type RIP1 and K45M-mutated RIP1 for expression in insects, and the constructs of RIP3 and RIP1 only with their respective catalytic domain for expression in E.coli. And we have thus expressed and obtained the corresponding proteins with high purity and activity. We have established the screening models for RIP3 inhibitors, and obtained 9 hits by screening 100 compounds available commercially. In addition, we have demonstrated the molecular mechanisms of action of the anticancer agent chimmitecan. Our present project is designed to answer the central question of whether RIP3 is suitable for an anticancer target, to identify its small-molecule inhibitors, and to explore their activities and molecular mechanisms of action.

抗肿瘤新靶点、新机制、新分子实体的发现与研究是创新型抗肿瘤药物研究研发的关键环节和核心基础。RIP3是丝/苏氨酸蛋白激酶，近年来发现其在决定细胞凋亡和坏死的死亡方式选择中发挥关键作用而广受关注。但其与肿瘤的关系、特别是能否作为合适的抗肿瘤靶点，迄今未被阐明，也无RIP3小分子抑制剂被报道。我们首次发现RIP3的表达显著抑制抗肿瘤药吉咪替康和SN38的作用，且可能独立于其决定细胞选择死亡方式的作用；还发现多种肿瘤细胞高表达RIP3。我们已建立RIP3及其底物RIP1和K45M突变型RIP1的全长真核表达体系，表达获得高活性高纯度的相应蛋白；建立分子细胞水平的RIP3抑制剂筛选体系，筛选并获得9个活性化合物；并阐明了吉咪替康的抗肿瘤作用机制。在此基础上，本课题将重点围绕RIP3是否能够作为合适的抗肿瘤靶点这一中心问题展开工作，并进行RIP3抑制剂的发现和其抗肿瘤/抗耐药作用及机制的系统研究。

RIP3是丝/苏氨酸蛋白激酶，近年来发现其在决定细胞凋亡和坏死的死亡方式选择中发挥关键作用而广受关注。但其与肿瘤的关系、特别是能否作为合适的抗肿瘤.靶点，迄今未被阐明，也无RIP3小分子抑制剂被报道。本课题通过研究RIP3与肿瘤和抗肿瘤药物之间的关系，发现并证明B-RafV600E抑制剂dabrafenib是高选择性强效RIP3抑制剂，在国际上第一次证明RIP3小分子抑制剂能够有效干预程序性坏死所致疾病，包括肿瘤和肝脏保护作用，并阐明其作用机制。申请并获授权发明专利1项、发表SCI论文4篇、培养研究生3名。本研究完成了原计划的各项工作、实现了预期的研究目标。