Small molecule inhibition of Rho GTPase activation to probe signaling cascades

小分子抑制 Rho GTPase 激活以探测信号级联

基本信息

批准号：
8535688
负责人：
JOHN E SONDEK
金额：
$ 28.19万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8535688
关键词：
Abnormal Cell Actins Adhesions Binding Biological Biological Assay Cell physiology Cells Chemicals Complex Computer Simulation Computing Methodologies Cytoskeleton Development Disease Dominant-Negative Mutation Environment Fluorescence Foundations Gene Expression Gene Silencing Generations Goals Growth Growth and Development function Guanine Nucleotide Exchange Factors Guanine Nucleotides Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Housing Human In Vitro Lead Libraries Link Malignant Neoplasms Malignant neoplasm of prostate Membrane Protein Traffic Methodology Methods Molecular Molecular Structure Monitor Nucleotides Phagocytosis Physiological Processes Physiology Process Proteins Protocols documentation Quantitative Structure-Activity Relationship Radioactive Regulation Research Resolution Resources Role Series Signal Pathway Signal Transduction Structure Structure-Activity Relationship Therapeutic Transcriptional Activation Transcriptional Regulation Validation assay development base cell growth cheminformatics cytotoxicity design drug development extracellular high throughput screening human disease improved inhibitor/antagonist malignant breast neoplasm migration novel therapeutics prevent programs response rho rho GTP-Binding Proteins screening small molecule stem tool

项目摘要

DESCRIPTION (provided by applicant): Activated Rho GTPases signal to numerous intracellular proteins to dynamically control vital cellular processes, including remodeling of the actin cytoskeleton, membrane trafficking, transcriptional regulation, cell growth, and development. The activation of Rho GTPases is catalyzed by Rho GEFs (guanine nucleotide exchange factors) in response to a variety of extracellular signals. This process is tightly controlled and spatially focused within cells, and abnormal regulation of several Rho GTPases is implicated in human cancers and other diseases. The long-term goal of this research is to more clearly define molecular mechanisms of Rho GTPase regulation in order to increase our understanding of Rho GTPase function in normal and pathological states and to improve our capacity to treat human diseases that stem from aberrant Rho GTPase signaling. The overall objective of this proposal is to develop a workflow comprised of an integrated set of assays for rapid identification and validation of compounds that can specifically and directly inhibit guanine nucleotide exchange and be used as probes of Rho GTPase function in cells. The goals of this proposal will be accomplished through three aims. In Specific Aim 1, a fluorescence-based nucleotide exchange assay will be optimized for high-throughput screening. In Specific Aim 2, high-throughput screening of diverse compound libraries will be implemented to identify inhibitors of P-Rex2-catalyzed activation of Rac1 and combined with structure similarity searching to improve the number and potency of initial hits. In Specific Aim 3, bona fide in vitro inhibition will be confirmed with a conventional radioactive-based exchange assay, and a series of in vitro and cell-based secondary assays will be developed to define the selectivity, cytotoxicity and biological activity of confirmed hits. The protocols developed here for Rac1 will be generally applicable to all Rho GTPases and are expected to yield lead compounds for generation of potent and selective probes of Rho GTPase function. These compounds may also serve as potential lead compounds for drug development.

描述（由申请人提供）：激活的Rho GTPases信号向许多细胞内蛋白质信号，以动态控制重要的细胞过程，包括重塑肌动蛋白细胞骨架，膜运输，转录调控，细胞生长和发育。 Rho GTPases的激活是由Rho GEFS（鸟嘌呤核苷酸交换因子）催化的，以响应各种细胞外信号。该过程受到严格控制，并在空间上集中在细胞内，并且对几种Rho GTPases的异常调节与人类癌症和其他疾病有关。这项研究的长期目标是更清楚地定义Rho GTPase调节的分子机制，以增强我们对正常和病理状态中Rho GTPase功能的理解，并提高我们治疗因异常Rho GTPase信号传导而治疗的人类疾病的能力。该建议的总体目的是开发一个工作流，该工作流程由一组集成的测定集组成，用于快速识别和验证化合物，这些化合物可以专门并直接抑制鸟嘌呤核苷酸交换并用作细胞中Rho GTPase功能的探针。该提案的目标将通过三个目标来实现。在特定的目标1中，将优化基于荧光的核苷酸交换测定法用于高通量筛选。在特定的目标2中，将实施对各种化合物库的高通量筛选，以识别RAC1的P-Rex2催化激活的抑制剂，并与结构相似性搜索相结合以提高初始点击的数量和效力。在特定的目标3中，将通过常规的基于放射性的交换测定法确认真正的体外抑制作用，并将开发一系列基于细胞和细胞的次要测定，以定义确认命中的选择性，细胞毒性和生物学活性。此处为Rac1开发的协议通常适用于所有Rho GTPase，并有望产生铅化合物，以生成Rho GTPase功能的有效探针和选择性探针。这些化合物也可能是药物开发的潜在铅化合物。