Chemical Modulation of the Siah-1 Pathway

Siah-1 通路的化学调节

• 批准号: 7694153
• 负责人: JOHN C REED
• 金额: $ 2.5万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7694153
• 关键词: Apoptosis; Binding; Binding Sites; Biological; Biological Assay; Biology; Breast Cancer Cell; Cancer cell line; Cell Cycle Arrest; Cell Cycle Regulation; Cell Line; Cell division; Cell physiology; Cells; Chemicals; Chemistry; Complex; Consensus; Cytokinesis; DNA Damage; Data; Degradation Pathway; Development; Engineering; Enzymes; Event; Excision; Family; Fibroblasts; Fluorescence Polarization; Genes; Goals; Human; Hypoxia; Laboratories; Libraries; Ligands; Link; Luciferases; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Messenger RNA; Methodology; Methods; Mitotic; Mixed Function Oxygenases; Mus; Mutation; N-terminal; Pathway interactions; Peptides; Plasmids; Play; Polyubiquitin; Post-Translational Protein Processing; Process; Production; Proline; Protein Family; Proteins; Proteolysis; Provider; Recovery; Reporter Genes; Reporting; Research; Role; Screening procedure; Serum; Siah-1 protein; Signal Pathway; Signal Transduction; Structure-Activity Relationship; TCF Transcription Factor; TP53 gene; Tertiary Protein Structure; Transcription Coactivator; Tubulin; Tumor Suppression; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; United States National Institutes of Health; Vav guanine-nucleotide exchange factor; Work; base; cell growth; cell transformation; cellular engineering; cheminformatics; high throughput screening; human NCOR1 protein; malignant stomach neoplasm; member; metabotropic glutamate receptor 4; multicatalytic endopeptidase complex; overexpression; pancreatic neoplasm; promoter; protein degradation; protein protein interaction; public health relevance; response; small molecule libraries; therapy resistant; tool; tumor; tumor growth; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：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 促进泛素转移至其底物蛋白，通过其 RING 结构域进行降解。 Siah-1 是高度保守的 RING 结构域蛋白家族的成员，可通过 β-catenin 降解途径调节多种细胞功能，包括细胞周期停滞、肿瘤抑制和细胞凋亡。 Siah-1 也被鉴定为 p53 诱导基因，在功能上将其与重要的肿瘤抑制因子联系起来。 Siah-1 通路的化学调节剂将为阐明该信号通路在癌症发生和进展中的作用提供强大的研究工具。在本提案中，我们描述了基于荧光偏振的高通量筛选（HTS）测定的开发，并利用带有附加荧光染料的 Siah 家族蛋白的肽配体。这种荧光偏振测定 (FPA) 构成了高通量竞争置换测定的基础，我们已针对化学库筛选进行了优化。我们建议使用这种 HTS 测定来筛选 NIH 化合物库。检测提供商提供的其他下游检测将针对去卷积命中进行。我们期望获得用于结构活性关系（SAR）研究的候选化合物，该研究将针对典型的包含 RING 的蛋白质 Siah-1 进行。总之，这些努力将产生经过验证的化学探针，用于研究各种生物环境中 Siah 家族 E3 连接酶的生物学。 公共健康相关性：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、肿瘤抑制和细胞凋亡。 Siah-1 通路的化学调节剂将为研究人类癌症的发生、进展和治疗耐药性通路提供强大的研究工具。