Chemical Modulation of the Siah-1 Pathway

描述（由申请方提供）：蛋白酶体降解通常需要用K48连接的多聚泛素链对靶蛋白进行翻译后修饰。这种蛋白质水解过程在正常细胞功能中起着关键作用。E3泛素连接酶Siah-1通过其RING结构域促进泛素转移至其底物蛋白以降解。Siah-1是高度保守的RING结构域蛋白家族的成员，其通过β-连环蛋白降解途径调节多种细胞功能，包括细胞周期阻滞、肿瘤抑制和凋亡。Siah-1也已被鉴定为p53诱导基因，在功能上将其与重要的肿瘤抑制因子连接。Siah-1通路的化学调节剂将为阐明该信号通路在癌症发展和进展中的作用提供强有力的研究工具。在该提案中，我们描述了基于荧光偏振的高通量筛选（HTS）测定的发展，并利用具有附着荧光染料的Siah家族蛋白的肽配体。这种荧光偏振分析（FPA）形成了高通量竞争性置换分析的基础，我们已经优化了化学库筛选。我们建议使用这种HTS测定来筛选NIH化合物库。将进行由测定提供商提供的其他下游测定，以解卷积命中。我们期望获得候选化合物的结构活性关系（SAR）的研究进行了一个原型环含蛋白质，Siah-1。总之，这些努力将导致验证化学探针，用于研究Siah家族E3连接酶在各种生物环境中的生物学。 公共卫生相关性：我们的目标是确定能够调节Siah-1蛋白酶体降解途径的化学物质。通过其作为泛素连接酶的活性，该信号通路有助于调节多种细胞事件，包括细胞周期停滞、肿瘤抑制和凋亡。Siah-1通路的化学调节剂将为研究人类癌症发展、进展和耐药途径提供强大的研究工具。

DESCRIPTION (provided by applicant): Proteasomal degradation typically requires post-translational modification of target proteins with K48-linked polyubiquitin chains. This process of protein proteolysis plays a key role in normal cellular function. The E3 ubiquitin ligase, Siah-1, facilitates the transfer of ubiquitin to its substrate proteins destined for degradation by way of its RING domain. Siah-1 is a member of a family of highly conserved RING domain proteins, which regulate a variety of cellular functions, including cell cycle arrest, tumor suppression, and apoptosis through the -catenin degradation pathway. Siah-1 has also been identified as a p53-inducible gene, functionally linking it to an important tumor suppressor. Chemical modulators of the Siah-1 pathway would provide powerful research tools for elucidating the roles of this signaling pathway in cancer development and progression. In this proposal, we describe the development of a High Throughput Screening (HTS) assay based upon fluorescence polarization, and utilizing a peptide ligand of Siah-family proteins with an attached flurochrome. 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 HTS assay. Additional downstream assays provided by the assay provider will be performed for deconvoluting hits. We expect to obtain candidate compounds for Structure Activity Relationship (SAR) studies to be performed for a prototypical RING- containing protein, Siah-1. Together, these efforts will result in validated chemical probes for studying the biology of Siah-family E3 ligases in a variety of biological settings. PUBLIC HEALTH RELEVANCE: Our goal is to identify chemicals capable of modulating the Siah-1 proteasomal degradation pathway. Through its activity as an ubiquitin ligase, this signaling pathway helps to regulate a variety of cellular events, including cell cycle arrests, tumor suppression, and apoptosis. Chemical modulators of the Siah-1 pathway will provide powerful research tools for studying pathways involved in human cancer development, progression, and resistance to therapy.