High-Throughput Screening for Modulators of Cytosolic Chaperonin Activity

描述（由申请人提供）：TCP-1环复合物（TRiC）是人类基因组编码的10-15%的蛋白质折叠和激活所必需的一个重要的，保守的伴侣。在其底物中有大量的细胞周期调节因子和肿瘤抑制蛋白。目前，没有特异性的激动剂或拮抗剂可用于细胞蛋白折叠的关键调节因子。鉴于其他伴侣导向抑制剂作为癌症治疗的早期前景，我们相信TRiC活性抑制剂也可能选择性地靶向转化细胞。在本项目中，我们计划：1)使用均匀时间分辨荧光（HTRF）方法对分子文库筛选中心网络化合物集合进行高通量筛选（HTS）鉴定TRiC的小分子抑制剂。2)测试HTS抑制剂对TRiC的特异性，并优化激活或抑制TRiC介导的肌动蛋白折叠的候选化合物。3)表征在组织培养中调节TRiC活性的分子的毒性和生物利用度，并测量候选药物在体内抑制蛋白质折叠的功效。

DESCRIPTION (provided by applicant): The TCP-1 Ring Complex (TRiC) is an essential, conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. Among its substrates are a large number of cell cycle regulators and tumor suppressor proteins. Currently, no specific agonists or antagonists are available for this key regulator of cellular protein folding. Given the early promise of other chaperone-directed inhibitors as cancer therapeutics, we believe that inhibitors of TRiC activity may also selectively target transformed cells. In this project, we plan to: 1) Identify small molecule inhibitors of TRiC with high throughput screening (HTS) using a homogenous time resolved fluorescence (HTRF) assay against the Molecular Libraries Screening Center Network compound collection. 2) Test the inhibitors from HTS for specificity to TRiC and optimize candidate compounds for activation or inhibition of TRiC-mediated actin folding. 3) Characterize the toxicity and bioavailability of molecules that modulate TRiC activity in tissue culture and measure the efficacy of candidates for inhibition of protein folding in vivo. PUBLIC HEALTH RELEVANCE: The chaperonin TRiC/CCT is essential for folding a large array of cellular proteins, including many cell cycle regulators, yet no inhibitors or modulators of its activity are available. Obtaining inhibitors for this chaperonin would be invaluable for understanding its biological function, and could also lead to a novel type of anti-cancer drugs.