蛋白翻译起始因子eIF4A的动态调控及化学干预研究

eIF4A is not only related to tumorigenesis and prognosis, but also the target of natural product, pateamine A, suggesting that eIF4AI is a potential drug target for tumor therapy. However, the internal catalytic mechanism of ATPase and RNA helicase of eIF4A remains unclear, which prevents the understanding of the biological function of eIF4AI and clinical application as a therapeutic target. In addition, all known inhibitors of eIF4AI were isolated from natural resources and their complex structures make them difficult to be synthesized for the structural optimization and future pre-clinical research. Our preliminary data implicate that the speed of Pi release will possibly determine the conformation change and coupling between ATPase and Helicase of eIF4AI. The binding sites of PatA were predicted through molecular docking and partially confirmed by the enzymatic assay in vitro. In this proposal, we will consequentially verify that Pi release is a key step to the conformation circulation of eIF4AI and eventually demonstrate the dynamically catalytic mechanism of eIF4AI. Moreover, we will screen for the novel chemical entities of the inhibitor of eIF4AI through the virtual screening or in vitro enzymatic screening in the light of the conclusion from the study of catalytic mechanism of eIF4AI. The final validated candidates will be utilized for the functional study of eIF4AI in vivo and its potential application in clinical will be explored.

eIF4A不但与肿瘤的发生发展和预后有着紧密的联系，而且与抗肿瘤候选化合物pateamine A在体内直接结合，是一个潜在的新的肿瘤治疗靶标。eIF4A本身的ATP水解酶和RNA解旋酶之间的动态催化机制尚不明确；以及现有的eIF4A的小分子配体均为天然产物，结构较为复杂，难以大量获得，这些因素妨碍了针对eIF4AI分子探针的结构优化，也限制了对eIF4A的生物功能的深入研究和后续的临床转化研究。申请人运用计算生物学结合药物设计、生物化学等研究手段，发现并验证磷酸的释放可能与eIF4AI两种酶活的偶联有关；初步预测和验证了PatA的结合位点。本项目拟在此前期研究基础上，进一步揭示eIF4AI两种酶活的偶联机制，阐明其动态催化机制；并针对eIF4AI功能循环中的不同构象状态，设计和筛选特异性的小分子探针，通过化学干预探究其生物功能和临床应用。

蛋白翻译起始因子eIF4AI是一个潜在的肿瘤治疗靶标，我们深入探讨了eIF4AI的ATP酶与RNA解旋酶的偶联机制，诠释了ATP水解的磷酸释放“门”控偶联机制。同时，还发现了Linker区域的一个关键突变体——eIF4AIL243G，它对已知的eIF4A抑制剂不敏感。.在此基础上，我们针对eIF4AI的ATP酶活性进行高通量筛选，筛选了课题组的活性天然小分子化合物库，以eIF4AIL243G为参考，最终发现了新型的eIF4AI小分子探针血根碱（Sanguinarine，SAN）。SAN是一个来源于罂粟科植物的活性天然小分子化合物，具有抗肿瘤、抗炎、抗微生物等多种生物学活性，然而其具体的直接作用靶标仍然不清楚。本研究在体外和体内确证了eIF4A是SAN的直接作用靶标，并鉴定到SAN作用于eIF4AI的N端结构域；通过晶体学技术，首次解析了eIF4AI-抑制剂复合体的共晶结构，分辨率达到原子级的1.31 Å，阐明了SAN与eIF4AI的结合口袋。通过结构对比发现SAN处于eIF4AI的N端和C端结构域的界面，能够阻止eIF4AI形成催化活性所必需的closed构象。我们还通过smFRET技术阐释了SAN的抑制机理，发现SAN阻止eIF4AI催化过程中各个构象的正常转化，导致semi-open构象累积，从而抑制酶活。.综上所述，本项目通过高通量筛选获得了新型的eIF4AI小分子探针SAN，首次阐明了eIF4AI位于N端结构域的一个特异性可靶向位点，进一步揭示了eIF4AI的酶活偶联及动态催化机制，为未来SAN的应用研究、eIF4AI的功能研究以及靶向eIF4AI的新药研发都提供了重要的理论依据。.在国自然基金资助下，本项目顺利完成研究目标和内容，发表SCI论文5篇，国内核心期刊1篇，参加国内会议2次（主题报告2次），毕业博士研究生3人，硕士研究生1人。