化学干预CBX4对低氧诱导因子1a的SUMO化修饰及其抗肿瘤效应

Post-translational modification is a key mechanism in regulating dynamic protein reactions and protein interactions. The applications of small-molecule compounds as probes have promoted our understanding on the regulating mechanisms of protein dynamic modifications and provided a new perspective for the study of protein functions and signal regulation pathways and for the development of innovative medicine. Hypoxic microenvironment is a key hallmark of tumor and the main transcriptional factor regulating the adaptive responses of tumor cells to hypoxia is hypoxia-inducible factor-1 (HIF-1). Previously, our group demonstrated that chromobox 4 (Cbx4 or Polycomb 2, Pc2) enhances the transcriptional activity of HIF-1a by its E3 ligase activity where it specifically sumoylates HIF-1a on K391 and K477. This results in elevated expressions of vascular endothelial growth factor (VEGF) and subsequent angiogenesis and metastasis of hepatocellular carcinoma, which was published in “Cancer cell”. Based on these studies, we have now developed a high-throughput and high-content screening system and screened 100,000 small chemical compounds for inhibitors that block the interaction between Cbx4 and HIF-1a or/and inhibit Cbx4 SUMO E3 activities. So far five active compounds have been identified and validated at the cellular level. In this project proposal, focusing on the key scientific theme of "chemical interventions of dynamic modifications of biological macro-molecules" highlighted in the funding guidance and leveraging active compounds as molecular probes, we plan to optimize the structures of these small-molecule compounds, and at the same time, validate the targets and mechanisms of these compounds through studies of chemical interventions of Cbx4 dynamic modifications of HIF-1a at levels of molecules, cells, tissues and animal models. This project aims to develop small-molecule chemical tools with high selectivity, specificity and bio-compatibility, reveal the regulatory mechanism of Cbx4 in sumoylating HIF-1a, and establish a link between the dynamic SUMO modification of HIF-1a and the discovery of drugs targets. Overall this project will validate the SUMO activities of Cbx4 as the target for the leading compounds and will provide basis for the development of new strategy and new compounds for the treatment of liver cancer.

应用小分子作为探针快速推动了对蛋白质动态修饰调控的认识，也为研究蛋白质功能、信号调控通路和药物研发提供了新策略和方法。我们报道Cbx4导致HIF-1a蛋白SUMO化修饰，显著增强其转录活性，促进肝癌新生血管形成和转移。在此基础上，我们建立高通量高内涵筛选体系，已经从10万种化合物中获得5个有效抑制Cbx4的SUMO化修饰活性和（或）抑制Cbx4与HIF-1a互作的活性化合物，并在细胞水平的初步验证。本课题拟以这些活性化合物为探针，从分子、细胞、组织和动物模型等层面开展Cbx4动态修饰HIF-1a的化学干预研究，确认相应的先导化合物的作用靶标及机制，并在优化化合物结构，分析其成药性的同时，发展高选择、高特异、高生物相容性的小分子化学工具，揭示Cbx4动态SUMO化修饰HIF-1a的调控机制，建立HIF-1a的SUMO化修饰与分子靶向药物发现之间的桥梁，为肿瘤的治疗提供新的理论基础和先导药物。

本项目在课题组已有的重要原创性发现的基础上，以针对Cbx4介导的HIF-1α转录活性的高通量高内涵体系筛选得到活性先导化合物，优化改造后得到探针分子并鉴定和确认其作用靶标及机制，并研究其体内外抗肝癌效应，进而发展高选择性、高特异性、高生物相容性的小分子化学工具，揭示Cbx4动态SUMO化修饰HIF-1的调控机制，建立HIF-1动态SUMO化修饰与分子靶向药物发现之间的桥梁。我们以HRE-Luciferase作为信号输出,建立双荧光素酶报告基因检测系统，进行高通量活性化合物的筛选。在所有筛选的10万种化合物中得到了5个不影响HIF-1本身的转录活性，但能够抑制Cbx4对HIF-1转录活性增强作用的化合物。其中一类命名为XZA化合物，我们发展得到了光亲和探针XZA-38，进行蛋白垂钓和质谱分析鉴定发现羟酰辅酶A脱氢酶（Hydroxyacyl-coenzyme Adehydrogenase, HADH）为化合物作用靶点，敲减后XZA化合物几乎不能抑制Cbx4对HIF-1转录活性的增强作用，高表达则抑制Cbx4对HIF-1转录活性的增强作用。HADH为脂肪酸β氧化通路的代谢酶，催化羟脂酰辅酶A氧化生成酮脂酰辅酶A。我们发现其上游底物并不影响HIF-1的SUMO化水平，而下游产物直接抑制了HIF-1的SUMO化修饰。进一步发现酮脂酰基团而非辅酶A部分起了决定性的作用，抑制了Cbx4的SUMO化活性。代谢组学实验也证实，化合物处理后的细胞，其胞内酮脂酰辅酶A明显增多。通过体外SUMO化反应体系，也证实了XZA-35本身无法抑制Cbx4的SUMO化活性，而是通过增加酮脂酰辅酶A的浓度抑制了Cbx4的SUMO化活性。通过蛋白质质谱，我们鉴定到了Cbx4蛋白的K106位点能够发生酮脂酰化修饰。我们制备了该位点特异性的酮脂酰化赖氨酸抗体，在细胞上验证了K106位赖氨酸酮脂酰化修饰可以被XZA-35调控，且该位点被酮脂酰化修饰后Cbx4的SUMO化活性被抑制，导致其底物HIF-1的SUMO化水平下降，下游VEGF基因表达受抑制。总之，我们首次发现了具有生物学功能的酮脂酰化修饰，并发展了小分子来动态调控其翻译后修饰过程，揭示了其在肿瘤发生发展中的作用，提出了潜在的抗肿瘤治疗策略。