【作者】 方勇；

【导师】 马丁；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 博士

【摘要】 目的：建立针对mTORC2特异性的下游靶点p-AKT (Ser473)的细胞分子模型,与华北制药新药研究开发公司合作,从微生物菌种代谢文库中筛选出mTORC2特异性的天然小分子抑制剂；方法：以mTORC2特异性的下游靶点p-AKT (Ser473)为第一轮的筛选靶点,分别以DMSO、Rapamycin、PI-103、HRP作为阴性对照、激活性阳性对照、抑制性阳性对照和底物空白对照,在正常的支气管上皮细胞系BEAS-2B中进行Cytoblot筛选。每种样品设计8种浓度梯度,各梯度之间呈3倍稀释。第一批检测样品为华北制药新药筛选中心拥有的1693种单体化合物；结果：将信号值低于PI-103孔或相当,重复性和浓度依赖性好的样品,进一步进行Western验证,获得3种对p-AKT (Ser473)有抑制活性的小分子抑制剂；将信号值高于Rapamycin或相当,重复性和浓度依赖性好的样品,同样进行Western验证,获得1种对p-AKT (Ser473)有激活作用的小分子激动剂；结论：针对p-AKT (Ser473)磷酸化水平的Cytoblot筛选方法,是一种基于活细胞整体,简单、快捷、灵敏度和特异性都比较高的方法,可以作为一种筛选mT0RC2特异性抑制剂的初筛模型；目的：初步探讨小分子化合物HA选择性诱导Rictor蛋白发生降解的分子机制，为筛选mTORC2特异性抑制剂提供一种新的思路；方法：Western进一步检测HA对多种肿瘤细胞中Rictor蛋白的影响；在HA诱导的Rictor蛋白降解的同时，加入蛋白酶体抑制剂MG-132，抑制蛋白酶体降解途径，发现Rictor蛋白的降解可以被逆转；将Rictor分成5段，分别构建GST融合蛋白，建立体外泛素模型，进一步确证Rictor蛋白可以被泛素化修饰；结果：HA可以选择性的诱导MM细胞中Rictor蛋白发生泛素．蛋白酶体途径降解；同时不会诱导mTOR蛋白和mTORC1复合体中的Raptor蛋白发生降解；在HA诱导的Rictor蛋白降解模型中，可以看到：Rictor蛋白被降解，mTORC2功能受抑，其特异性的底物p-AKT(Ser473)位点磷酸化水平降低；加用蛋白酶体抑制剂时，逆转Rictor降解，p-AKT(Ser473)位点磷酸化水平得以恢复；体外泛素模型中，发现Rictor蛋白可以被泛素化修饰；结论：基于HA诱导Rictor蛋白的降解现象，深入研究选择性降解Rictor蛋白的分子机制，将为筛选mTORC2特异性抑制剂提供一种全新的策略。更多还原

【Abstract】 Objective:To establish the cellular and molecular models for phosphorylation-AKT (Ser473) which is the specific downstream target of mTORC2, cooperating with North China Pharmaceutical Group (NCPC) New Drug Research and Development Co.Ltd, and screening specific small molecule inhibitor of mTORC2 from the microbial strain metabolism library;Methods:We used phosphorylation-AKT (Ser473) which was the specific downstream target of mTORC2 as the screening targets of first round. Using DMSO, Rapamycin, PI-103, HRP as negative control, activation positive control, suppression positive control and substrate blank control respectively, we conducted Cytoblot screening in normal bronchial epithelial cell line BEAS-2B. Eight concentration gradients were designed in each sample,3-fold dilution between two adjacent gradients. The first test samples were 1693 kinds of monomeric compounds owned by North China Pharmaceutical Group (NCPC) Center for New Drug Screening;Results:When the signal value of a compound was lower than the value of PI-103 or equivalent, and presenting preferable reproducibility and concentration-dependent manner, we took further Western identification, and finally obtained three kinds of small molecule inhibitors which had suppressive activity of phosphorylation-AKT (Ser473); When the signal value of a compound was higher than the value Rapamycin or equivalent, and also presenting preferable reproducibility and concentration-dependent manner, we took further Western identification, and finally obtained one kind of small molecule agonist which had activation on phosphorylation-AKT (Ser473).Conclusions:Phosphorylation-AKT (Ser473) Cytoblot screening method, is based on the whole living cells, which is simple and fast, having high sensitivity and specificity. It’s could be used as a preliminary screening model to screen specific inhibitors of mTORC2; Objective: To explore molecular mechanism of the phenomenon that small moleculecompounds HA induced Rictor protein degradation selectivly, and providing a novel threadfor screening specific inhibitors of mTORC2 complex;Methods: We further tested the effects of HA induced Rictor protein degradation in avariety of tumor cell lines by western blot; in HA-induced Rictor protein degradation model,when we added the proteasome inhibitor MG-132, which could inhibit the proteasomedegradation pathway, Rictor protein degradation could be reversed; and then we dividedRictor into five clips, for the Rictor protein is too large, to reconstruct GST fusion proteinsrespectively, established In vitro Ubiquitination model, further confirmed Rictor proteincould be ubiquitylated;Results: HA could selectively induce Rictor protein degradation occurring on MM cellline in ubiquitin-proteasome pathway; at the same time, HA could not induce mTORprotein and Raptor protein degradation, both of which are the important constituents of thecomplex of mTORC1; in HA-induced Rictor protein degradation model, we could see thatRictor protein was degraded, mTORC2 function was inhibited, the phosphorylation level ofits specific substrate p-AKT (Ser473) sites reduced; when combining with proteaseinhibitor, the Rictor protein degradation was reversed, the phosphorylation level of p-AKT(Ser473) sites was recovered;Conclusion: Based on the induced Rictor degradation model by the compound of HA, bythorough research the molecular mechanism of this selective degradation, we will get apromising strategy to screen specific inhibitors of mTORC2.更多还原