Chemical Antagonists of IAP-Family Anti-Apoptotic Proteins

描述(由申请人提供)：细胞凋亡在正常发育和生理的许多方面起着重要的作用，在以细胞死亡不足或过度为特征的无数疾病中变得失调。半胱氨酸氨基转移酶是细胞凋亡的执行者。这些细胞内的蛋白酶被凋亡抑制蛋白(IAPs)抑制，IAPs是一类进化上保守的抗凋亡蛋白。线粒体释放的蛋白质(SMAC和HtrA2)可以竞争性地取代Caspase中的IAP，从而帮助推动细胞凋亡。研究表明，只有激活的Smac蛋白(4聚体)N-末端的少数残基足以影响Caspase释放IAP。因此，通过使IAP释放Caspase来模拟Smac拮抗IAP的作用的化合物是可信的。非肽基化学抑制剂在细胞通透性、稳定性和体内药理方面均优于Smac多肽。为此，我们开发了一种基于荧光偏振的结合分析方法，使用一个代表激活的Smac N-末端残基的短肽与一个连接的荧光色素结合。这种荧光偏振分析(FPA)形成了高通量竞争置换分析的基础，我们已经为化学文库筛选进行了优化。我们建议使用这个FPA来筛选NIH化合物文库，从而识别与Smac肽竞争结合IAP的化合物。然后，使用我们已经设计的三种二次分析方法，将独立地确认命中。类似物的结构活性关系(SAR)研究将针对IAP-家族的原型成员XIAP进行。最后，为了确定化合物的选择性，将使用为IAP家族的其他成员(cIAP1、cIAP2、ML-IAP、ILP2)配置的分析来进行SAR研究。总之，这些努力将导致有效的化学探针，用于在各种细胞和生物背景下研究IAP的生物学。细胞死亡是生理学的一个正常方面。人类平均产生并同时根除体内500-700亿个细胞，其中大部分细胞死亡是通过一个被称为“细胞凋亡”的过程发生的。细胞凋亡正常调控的缺陷是许多疾病的核心，包括癌症(细胞死亡不足导致细胞异常聚集)，以及退行性疾病(细胞过度死亡导致组织丢失和器官功能障碍)。细胞内的蛋白水解酶，称为半胱氨酸天冬氨酸氨基转移酶，来完成细胞的凋亡。像所有的蛋白分解系统一样，Caspase受到蛋白质网络的精细控制，这些蛋白质网络要么促进它们的激活，要么抑制它们的活性。Caspase的主要内源性拮抗剂是IAPs，这是一个进化上保守的蛋白家族，与Caspase结合并抑制其活性，或通过泛素依赖的机制诱导Caspase降解。我们的目标是确定与IAP结合位点的化合物，竞争性地取代Caspase。所得到的化合物将作为研究工具，用于理解IAP的生物学，并确定它们在癌症等疾病中的作用，在癌症中，IAP的过度表达是常见的。

DESCRIPTION (provided by applicant): Apoptosis plays essential roles in many aspects of normal development and physiology, becoming dysregulated in myriad diseases characterized by insufficient or excessive cell death. Caspases are the executioners of apoptosis. These intracellular proteases are suppressed by Inhibitor of Apoptosis Proteins (IAPs), a family of evolutionarily conserved anti-apoptotic proteins. Proteins released from mitochondria (SMAC and HtrA2) can competitively displace IAPs from the Caspases, thus helping to drive apoptosis. It has been shown that only a few residues at the N-terminus of activated SMAC protein (4mer) are sufficient to affect the release of IAPs from Caspases. Thus, it is plausible to identify chemical compounds that mimic the effect of SMAC in antagonizing IAPs by causing them to release Caspases. Non-peptidyl chemical inhibitors would have advantages over SMAC peptides, in terms of cell permeability, stability, and in vivo pharmacology. To this end we have developed a binding assay based upon fluorescence polarization, using a short peptide representing residues from the N-terminus of activated SMAC with an attached fluorochrome. This fluorescence polarization assay (FPA) forms the basis for a high-throughput competitive displacement assay that we have optimized for chemical library screening. We propose to screen the NIH compound library using this FPA and thus identify chemical compounds that compete with SMAC peptide for binding to IAPs. Then, using 3 types of secondary assays we have already devised, the hits will be independently confirmed. Structure Activity Relations (SAR) studies of analogs will be performed for a prototypical member of the IAP-family, XIAP. Finally, to define the selectivity of the compounds, SAR studies will be performed using assays configured for additional members of the IAP family (cIAP1, cIAP2, ML-IAP, ILP2). Altogether, these efforts will result in validated chemical probes for studying the biology of IAPs in a variety of cellular and organismal contexts. Cell death is a normal facet of physiology. The average human produces and in parallel eradicates 50-70 billion cells in his or her body, with most of this cell death occurring via a process known as "apoptosis." Defects in the normal regulation of apoptosis are at the core of many diseases, including cancer where insufficient cell death permits abnormal cell accumulation, and degenerative diseases where excessive cell death leads to tissue loss and organ dysfunction. Apoptosis is accomplished by intracellular proteases, called Caspases. Like all proteolytic systems, the Caspases are under fine control by networks of proteins that either promoter their activation or suppress their activity. The chief endogenous antagonists of Caspases are IAPs (Inhibitor of Apoptosis Proteins), an evolutionarily conserved family of proteins that bind to and inhibit the activity of Caspases or that induce Caspase degradation by ubiquitin-dependent mechanisms. Our objective is to identify chemical compounds that bind sites on IAPs, competitively displacing Caspases. The resulting compounds will be useful as research tools for understanding the biology of IAPs and for ascertaining their roles in diseases such as cancer, where IAP over-expression is commonly observed.