HTP assays of inhibitors of protein-protein interactions

热休克蛋白90 （Hsp90）是一种“分子伴侣”，其活性对许多促进癌细胞存活和增殖的突变、嵌合或过表达蛋白的折叠和细胞稳定性至关重要。其活性依赖于hsp90的蛋白包括突变的p53、Brc-Abl、Raf-1、Src和ErbB2。抑制HspQO活性是广泛的抗癌药物的潜在途径。格尔达霉素是一种抑制热休克蛋白90活性的小分子，目前正作为一种潜在的抗癌药物进行临床试验。Hsp90并不是孤立地起作用的，而是一个复杂机制的一部分，该机制涉及其他几种蛋白质和“合作伙伴”。两种伴侣蛋白，Hsp70和Hsp90，依次作用于特定的客户蛋白，产生成熟的、活性的蛋白形式。Hsp组织

Heat shock protein 90 (Hsp90) is a "molecular chaperone" whose activity is essential for the folding and cellular stability of a number of mutated, chimeric or over-expressed proteins that promote the survival andproliferation of cancer cells. Examples of proteins whose activity is Hsp90-dependent include mutated p53, Brc-Abl, Raf-1, Src, and ErbB2. Inhibition of HspQO activity is a potential route to broad range anti-cancer agents. Geldanamycin, a small molecule that inhibits Hsp90 activity, is currently in clinical trials as a potential anti-cancer agent. Hsp90 does not function in isolation, but rather is part of a complex machinery that involves several other proteins and "co-chaperpnes". Two chaperones, Hsp70 and Hsp90, act sequentially on certain client proteins to generate the mature, active, form of the proteins. Hsp Organizing Protein (HOP) has independent binding sites for Hsp70 and Hsp90, and by binding these chaperones simultaneously, it functions to bring them into physical proximity. The interaction of HOP with Hsp70 and Hsp90 is very well characterized. HOP has two independent tetratricopeptide repeats (TRP). TPR1 binds specifically to the C-terminal peptide of Hsp70 and TPR2A binds specifically to the C-terminal peptide ofHsp90. We propose to develop HTP assays to identify specific inhibitors of the interaction of Hsp 90 withTPR2A. Inhibition of this interaction will prevent the Hsp70-Hsp90 dependent folding sequence, a nd thus lead to oncogene degradation. It has already been demonstrated in mammalian cells that preventing its interaction with HOP inhibits Hsp90 activity. We will develop fluorescent-based assays, in vitro and in vivo to identify small molecules that specifically disrupt the Hsp90-HOP interaction. We will follow-up potential leads by assaying their ability to reverse the oncogenic phenotype in HTP morphology assays. Promising compounds will go forward into trials in collaboration with other researchers.