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HTP assays of inhibitors of protein-protein interactions

蛋白质-蛋白质相互作用抑制剂的 HTP 测定

基本信息

批准号：
7196409
负责人：
LYNNE J. REGAN
金额：
$ 31.53万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-15 至 2008-03-31
项目状态：
已结题

项目摘要

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.

热休克蛋白90（Heat shock protein 90，Hsp 90）是一种分子伴侣，其活性对许多突变、嵌合或过表达的蛋白质的折叠和细胞稳定性至关重要，这些蛋白质促进癌细胞的存活和增殖。其活性是Hsp 90依赖性的蛋白质的实例包括突变的p53、Brc-Abl、Raf-1、Src和ErbB 2。HspQO活性的抑制是获得广泛抗癌剂的潜在途径。格尔德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。热休克蛋白90并不是孤立地发挥作用，而是一个复杂机制的一部分，该机制涉及几种其他蛋白质和“辅助伴侣”。两种分子伴侣Hsp 70和Hsp 90依次作用于某些客户蛋白质以产生成熟的活性形式的蛋白质。Hsp组织蛋白（HOP）对Hsp 70和Hsp 90具有独立的结合位点，并且通过同时结合这些分子伴侣，它的功能是使它们在物理上接近。HOP与Hsp 70和Hsp 90的相互作用被很好地表征。HOP具有两个独立的三肽重复序列（TRP）。TPR 1特异性结合Hsp 70的C-末端肽，TPR 2A特异性结合Hsp 90的C-末端肽。我们建议发展HTP检测来鉴定Hsp 90与TPR 2A相互作用的特异性抑制剂。抑制这种相互作用将阻止Hsp 70-Hsp 90依赖性折叠序列，从而导致癌基因降解。已经在哺乳动物细胞中证明，阻止其与HOP的相互作用抑制Hsp 90活性。我们将在体外和体内开发基于荧光的检测方法以鉴定特异性破坏Hsp 90-HOP相互作用的小分子。我们将通过在HTP形态学试验中测定其逆转致癌表型的能力来跟踪潜在的线索。有希望的化合物将与其他研究人员合作进行试验。