INHIBITORS OF THE RAS-RAF-MAP KINASE SIGNALLING PATHWAY

该实验室计划的重点是识别抑制剂， 参与raf-1早期事件的丝氨酸/苏氨酸蛋白激酶 MAP激酶通路。由于这些普遍存在的激酶是 参与调节细胞生长和分化的信号传导， 预期这种抑制剂对 肿瘤细胞表型这些激酶，它们相互激活， 一个级联途径，信号下游致癌p21 ras蛋白， 以及蛋白激酶C（PKC）和酪氨酸蛋白的下游 激酶（TPKs）。该途径的作用是磷酸化， 激活参与基因转录和翻译的蛋白质。这 该项目是从之前的PKC调节重点发展而来的 融资期。焦点的转移使得最近的知识 raf-MEK-MAPK信号转导的发现，以及所做的工作， 实验室项目负责人获得的经验， 靶向这一重要的信号通路， 过程本项目的具体目标是：（1）确定 p74 raf-1、MEK和ERK/MAP激酶的抑制剂;（2）表征 这些抑制剂的特异性;和（3）确定这些抑制剂的功效。 阻断p74 raf-1、MEK或ERK/MAPK活性的抑制剂， 细胞，包括急性有丝分裂反应和正常和 转化细胞萃取物、分馏、化合物和结构 项目1将进行说明。raf-MEK-的筛选方法 MAPK通路抑制剂在这个实验室计划是类似的， PKC，使用纯化的重组酶和基于放射性的96孔 比色法然而，这些途径激酶及其信号传导作用是 比PKC更新发现， 随着更多的发现，预计将进行二次化验。过去 经验表明，提取物分级的优先顺序 应将低优先级分配给含有抑制活性的样品 可能由多酚、醌类和类黄酮引起， 常见但无趣的蛋白激酶抑制剂。的其他因素 用于提取物分级分离增加的优先级分配包括高 效力、可用性和高靶特异性。测试 分离的抑制剂的特异性包括检测PKC的抑制 和PKA在这个实验室计划，除了分析提供了由 项目2和4。急性体外细胞试验在本实验室进行 程序和细胞毒性测试以及体内功效测定由 分别是核心B和C。这些激酶的抑制剂将是独特的 和有用的工具，在信号转导研究和生化活性 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药物。

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.