INHIBITORS OF THE RAS-RAF-MAP KINASE SIGNALLING PATHWAY

RAS-RAF-MAP 激酶信号通路抑制剂

基本信息

批准号：
6300370
负责人：
CURTIS L. ASHENDEL
金额：
$ 16.56万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-05-01 至 2000-08-31
项目状态：
已结题

项目摘要

The focus of this laboratory program is identification of inhibitors of serine/threonine protein kinases involved in early events in the raf-1 to MAP kinase pathway. Since these ubiquitous kinases are centrally involved in signaling that regulates cell growth and differentiation, such inhibitors are expected to have significant effects on the neoplastic cell phenotype. These kinases, which activate each other in a cascading pathway, signal downstream from oncogenic p21ras protein as well as downstream from protein kinase C (PKC) and tyrosine protein kinases (TPKs). The effect of this pathway is to phosphorylate and activate proteins involved in gene transcription and translation. This project evolved from a focus on modulation of PKC during the previous funding period. The shift in focus allows knowledge from recent discoveries in raf-MEK-MAPK signaling, as well as work done and experienced gained by the lab program leader to be applied to the targeting of this important signaling pathway for the drug discovery process. The specific goals of this projects are to: (1) identify inhibitors of p74raf-1, MEK, and ERK/MAP kinases; (2) characterize the specificity of these inhibitors; and (3) determine the efficacy of these inhibitors for blocking the activity of p74raf-1, MEK, or ERK/MAPK in cells, including acute mitogenic responses and growth of normal and transformed cells. Extracts, fractionation, compounds, and structure elucidation will be done by project 1. The screening method for raf-MEK- MAPK pathway inhibitors in this lab program is similar to that done for PKC, using purified recombinant enzymes and a radioactivity-based 96-well assay. However, these pathway kinases and their signaling roles were discovered more recently than PKC and changes in the screening and secondary assays are anticipated as more discoveries are made. Past experience indicates that prioritization of extracts for fractionation should assign low priority to samples containing inhibitory activity potentially caused by polyphenols, quinones, and flavinoids, which are common but uninteresting protein kinase inhibitors. Other factors that increase priority assignment for fractionation of extracts include high potency, availability, and high target specificity. Testing the specificity of isolated inhibitors includes testing for inhibition of PKC and PKA in this lab program, in addition to the assays are provided by projects 2 and 4. Acute in vitro cellular assays are done in this lab program and cytotoxicity testing and in vivo efficacy assays are done by Cores B and C, respectively. Inhibitors of these kinases will be unique and useful tools in signal transduction research and biochemically active compounds that are efficacious in cellular and whole animal rodent models will be candidates for preclinical development.

该实验室计划的重点是鉴定抑制剂丝氨酸/苏氨酸蛋白激酶参与 raf-1 的早期事件 MAP 激酶途径。由于这些普遍存在的激酶集中在参与调节细胞生长和分化的信号传导，预计此类抑制剂将对肿瘤细胞表型。这些激酶在以下过程中相互激活：级联途径，致癌 p21ras 蛋白下游的信号以及蛋白激酶 C (PKC) 和酪氨酸蛋白的下游激酶（TPK）。该途径的作用是磷酸化和激活参与基因转录和翻译的蛋白质。这该项目是从之前对 PKC 调制的关注发展而来的资助期限。焦点的转变允许从最近的知识 raf-MEK-MAPK 信号传导方面的发现以及已完成的工作和实验室项目负责人获得的经验可应用于针对药物发现的这一重要信号通路过程。该项目的具体目标是：（1）确定 p74raf-1、MEK 和 ERK/MAP 激酶抑制剂； (2) 表征这些抑制剂的特异性； (3) 确定这些措施的功效用于阻断 p74raf-1、MEK 或 ERK/MAPK 活性的抑制剂细胞，包括急性有丝分裂反应以及正常和正常细胞的生长转化的细胞。提取物、分馏、化合物和结构阐明将由项目1完成。raf-MEK-的筛选方法该实验室项目中的 MAPK 通路抑制剂与 PKC，使用纯化的重组酶和基于放射性的 96 孔板化验。然而，这些途径激酶及其信号传导作用是比 PKC 更晚发现，并且筛选和检测方面发生了变化随着更多发现的出现，预计将进行二次测定。过去的经验表明，分馏提取物的优先顺序应为含有抑制活性的样品分配低优先级可能是由多酚、醌和类黄酮引起的，这些物质常见但无趣的蛋白激酶抑制剂。其他因素增加提取物分级的优先级分配，包括高效力、可用性和高目标特异性。测试分离抑制剂的特异性包括 PKC 抑制测试和 PKA 在此实验室计划中，除了由项目 2 和 4。急性体外细胞测定在本实验室进行程序和细胞毒性测试以及体内功效测定是由分别为核心 B 和 C。这些激酶的抑制剂将是独特的以及信号转导研究和生化活性方面的有用工具在细胞和整个动物啮齿动物模型中有效的化合物将成为临床前开发的候选者。