基于三维结构的睾丸孤核受体4抑制剂的筛选及抑制机制研究

Recent studies show that TR4 over-expression is positively correlated to the phenotype of corticotroph tumors induced Cushing Disease as well as to the treatment-resistance in prostate cancer. Therefore, it is of importance to develop highly effective TR4 inhibitors. It has been proved that the dimerization of TR4 is necessary for its transactivation activity. Through detailed analysis of TR4-LBD crystal structure, we found a groove area at the dimer interface area in which several key residues such as Arg-570, Leu-563, Asp-546 are located. In this study, we define the above described groove area as a binding pocket for virtual screening of TR4 inhibitor lead compounds. The inhibition activities of the resulting compounds are tested via the established dual luciferase reporter assay. Furthermore, the mechanism of highly effective inhibitors will be further investigated at molecular, atomic and thermodynamic levels via native-PAGE, protein crystallization and ITC techniques. This study aims to screen and obtain highly effective TR4 inhibitors and provides structural basis for development of new drugs and sensitization agents in treatment of Cushing disease and prostate cancer, respectively.

近期研究表明睾丸孤核受体4(TR4)高表达与垂体腺瘤所致的库欣病表型及前列腺癌放化疗抵抗等呈正相关。目前亟待研究开发高效的TR4抑制剂。研究证实TR4二聚体形成是其转录激活所必需的。通过TR4-LBD晶体结构二聚体界面分析，我们发现一个由Arg-570，Leu-563，Asp-546等十多个关键氨基酸残基组成的凹槽区域，可以作为二聚体阻断剂的结合口袋。本课题拟针对上述空腔区域进行虚拟筛选，得到靶向TR4二聚体相互作用界面的TR4抑制剂先导化合物，利用双荧光素酶报告基因系统对筛选到的化合物进行生物活性测试，并通过非变性聚丙烯酰胺凝胶电泳、TR4-LBD抑制剂复合物晶体结构分析及等温滴定量热法等技术进一步从分子、原子及热力学水平阐明抑制剂的作用机理。此研究旨在发现TR4高效抑制剂，为寻找治疗库欣病的新药及前列腺癌治疗增敏剂提供理论基础。

近年来研究表明睾丸孤核受体4（TR4）促进泌尿生殖系统肿瘤的转移及治疗抵抗，是抑制前列腺癌、肾癌和精原细胞瘤转移以及治疗增敏的潜在靶点。本研究综合运用基于TR4三维结构的虚拟筛选和表面等离子共振芯片技术寻找TR4抑制剂的候选化合物，建立并优化了TR4抑制剂生物活性测试的双荧光素酶报告基因系统，最终发现了30余个对TR4具有抑制作用的化合物。随后，我们选取化合物bexarotene，对其抑制作用及相应机制做进一步研究。免疫荧光结果显示bexarotene可以减少TR4的胞核分布，增加TR4的胞浆分布；虚拟筛选结果提示bexarotene可能结合于TR4的配体结合口袋，表面等离子共振分析提示bexarotene可以直接与TR4配体结合结构域(TR4-LBD)相结合；圆二色谱结果显示bexarotene与TR4-LBD结合后可诱导TR4-LBD产生构象变化。目前bexarotene在临床上主要治疗皮肤T细胞淋巴瘤。我们的研究结果提示bexarotene有望抑制泌尿生殖系统肿瘤的侵袭转移，并有望与化疗药物联用提高药物疗效。本项目共计发表学术论文4篇，其中SCI期刊论文3篇，国内学术期刊1篇；利用本项目相关研究成果，直接获得国家基金面上项目资助1项，申请国家发明专利1项。直接或间接培养博士研究生2名，硕士研究生3名。