INHIBITORS OF THE RAS-RAF-MAP KINASE SIGNALLING PATHWAY

这项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 绘制蛋白激酶途径图。因为这些无处不在的激酶集中在 参与调节细胞生长和分化的信号， 这类抑制剂预计将对 肿瘤细胞表型。这些在体内相互激活的激酶 致癌p21ras蛋白AS下游的级联信号通路 以及蛋白激酶C(PKC)和酪氨酸蛋白的下游 蛋白水解酶(TPKs)。这个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 该项目从上一阶段对PKC调制的关注演变而来 资助期。焦点的转移使最近的知识 在RAF-MEK-MAPK信号方面的发现，以及所做的工作和 实验室项目负责人获得的经验将应用于 靶向这一重要的信号通路用于药物发现 进程。该项目的具体目标是：(1)确定 P74raf-1、MEK和ERK/MAP激酶的抑制剂；(2)表征 这些抑制剂的特异性；以及(3)决定这些药物的疗效 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和正常和 转化的细胞。萃取物、分级、化合物和结构 将通过项目1进行澄清。RAF-MEK的筛选方法- 本实验计划中的MAPK途径抑制剂类似于 使用纯化的重组酶和基于放射性的96孔 化验。然而，这些途径激酶及其信号转导作用是 比PKC发现得更晚，在筛查和 随着更多的发现，二次化验有望实现。过去时 经验表明，分级抽提物的优先顺序 应对含有抑制活性的样品给予较低的优先级 可能是由多酚、苯二酚和类黄素引起的，它们是 常见但乏味的蛋白激酶抑制剂。其他因素 增加萃取物分级优先级分配包括高 效力、可用性和较高的靶标特异性。测试 分离的抑制剂的特异性包括对PKC的抑制试验 和PKA，在本实验计划中，除了由 项目2和4.急性体外细胞分析是在这个实验室进行的 程序和细胞毒性测试以及体内疗效分析由 分别是B和C核。这些激酶的抑制剂是独一无二的 以及在信号转导研究和生化活性方面的有用工具 对细胞和全动物啮齿动物模型有效的化合物 将是临床前开发的候选者。

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.