Chemical Probes of Protein Prenylation

描述(由申请人提供)：蛋白质预烯基化是一个极其重要的翻译后修饰过程。它是许多信号转导蛋白，包括ras癌基因产物，正确的膜结合和活性所必需的。蛋白质法尼基转移酶(FTase)抑制剂作为抗癌药物的开发已引起人们的浓厚兴趣，并在临床试验中观察到了可喜的结果。然而，现在清楚的是，RAS不是FTase抑制剂的唯一目标，甚至不是最重要的目标。这项研究项目的长期目标是开发用于选择性调节蛋白质预烯基化的化学工具。这些探针将允许确定各种前烯基化蛋白在癌细胞生长中的相对作用。这一建议将解决以下具体假设：FTase的独特结构和机制允许发现选择性抑制剂，这些抑制剂将阻止某些蛋白质的预烯基化，但不会干扰其他蛋白质的预烯基化。我们将利用现有的和新合成的化学工具，结合新的生化和生物学合作研究，来解决以下具体目标：目的1)表征FTase和GGTase I中肽和异戊二烯选择性的相互作用。这些研究将使用现有的和新合成的异戊二烯类似物、简明的靶向合成多肽库和各种生物分析方法。将采用合理的设计和筛选方法来建立有效的选择性模式。目的2)利用现有的动力学技术，在分子水平上阐明FTase选择性戊烯基化的相关事件。详细的稳态和稳态前动力学实验，结合各种结合常数的测定，将被采用。这些研究将为FTase使用的机制提供新的见解，并为设计新的选择性FPP类似物提供见解。目的3)通过对RAS和其他异戊二烯基化蛋白的戊烯基异构体的直接定量，确定异戊二烯类似物在细胞内改变FT酶底物选择性的能力。还将确定法尼醇类似物对甲氧戊酸途径和胞内戊烯基二磷酸盐池通量的影响。这些目标的实现将为未来的研究提供工具和奠定基础，以探索该项目的长期假设：在蛋白质中选择性地引入修饰的戊烯基团将改变其亚细胞定位和生物活性。

DESCRIPTION (provided by applicant): Protein prenylation is a critically important post-translational modification process. It is required for the proper membrane association and activity of many signal transduction proteins, including the Ras oncogene products. There has been intense interest in the development of protein farnesyltransferase (FTase) inhibitors as anti-cancer agents, and promising results have been observed with these compounds in clinical trials. However, it is now clear that Ras is not the sole, or even the most important target of FTase inhibitors. The long-term goal of this research project is the development of chemical tools for the selective modulation of protein prenylation. These probes will allow for the determination of the relative roles of various prenylated proteins in cancer cell growth. This proposal will address the following specific hypothesis: The unique structure and mechanism of FTase allows for the discovery of selective inhibitors that will block the prenylation of certain proteins but not interfere with the prenylation of others. We will use currently existing and newly synthesized chemical tools, in combination with new biochemical and biological collaborative studies, to address the following specific aims: Aim 1) To characterize the interplay of peptide and isoprenoid selectivity in FTase and GGTase I. These studies will use existing and newly synthesized isoprenoid analogs, concise targeted synthetic peptide libraries, and various bioanalytical methods. Rational design will be employed along with screening methods to establish effective selectivity patterns. Aim 2) To elucidate the events covering selective prenylation by FTase at a molecular level, using existing kinetic techniques. Detailed steady state and pre-steady state kinetic experiments, in combination with various binding constant determinations, will be employed. These studies will provide novel insight into the mechanism employed by FTase, and insight into the design of new selective FPP analogs. Aim 3) To establish the ability of isoprenoid analogs to alter FTase substrate selectivity in cells, through the direct quantitation of prenyl isoforms of Ras and other prenylated proteins. The effects of farnesol analogs on flux through the mevalonate pathway and intracellularprenyl diphosphate pools also will be determined. The accomplishment of these aims will provide the tools and lay the groundwork for future studies to investigate the long-term hypothesis of this project: the selective introduction of modified prenyl groups into proteins will alter their subcellular localization and biological activity.