Small molecule inhibition of Rho GTPase activation to probe signaling cascades

描述（由申请人提供）：活化的Rho GTP酶向许多细胞内蛋白发出信号，以动态控制重要的细胞过程，包括细胞的重塑。 肌动蛋白细胞骨架，膜运输，转录调控，细胞生长和发育。Rho GTP酶的激活由Rho GEF（鸟嘌呤核苷酸交换因子）响应于多种细胞外信号而催化。这一过程在细胞内受到严格控制和空间聚焦，并且几种Rho GTP酶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 GT3调节的分子机制，以增加我们对正常和病理状态下Rho GT3功能的理解，并提高我们治疗源于异常Rho GT3信号传导的人类疾病的能力。该提案的总体目标是开发一个工作流程，该工作流程由一套完整的检测方法组成，用于快速鉴定和验证能够特异性和直接抑制鸟嘌呤的化合物 核苷酸交换并用作细胞中Rho GTPase功能的探针。本提案的目标将通过三个目标来实现。在特定目标1中，将优化基于荧光的核苷酸交换测定以进行高通量筛选。在具体目标2中，将对不同化合物文库进行高通量筛选，以鉴定P-Rex 2催化Rac 1活化的抑制剂，并结合结构相似性搜索，以提高初始命中的数量和效力。在特定目标3中，将使用常规的基于放射性的交换试验确认真正的体外抑制，并将开发一系列体外和基于细胞的二次试验，以确定确认命中的选择性、细胞毒性和生物活性。这里开发的Rac 1的协议将普遍适用于所有的Rho GTP酶，并预计产生的先导化合物产生的有效和选择性的探针Rho GTP酶功能。这些化合物也可以作为药物开发的潜在先导化合物。

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.