Inhibition of Prenylated Protein Processing

描述（由申请人提供）：与许多人类癌症有关的Ras家族成员在翻译后被修饰，将其靶向到适当的细胞内位置。Ras和其他caax蛋白经历3个连续的反应：半胱氨酸的异戊二烯化，特别是法尼基转移酶（FTase）的法尼基化；3末端氨基酸（-aaX）的蛋白水解；异戊二甲基化半胱氨酸的a-羧基甲基化。这个过程对于膜定位和关键Ras癌蛋白的活性至关重要。FTase抑制剂正在临床试验中作为癌症化疗药物进行评估。不幸的是，这些化合物对许多ras转化的肿瘤几乎没有作用。现在人们对随后的酶促步骤越来越感兴趣；Ras转换酶（Reel）的蛋白水解和异戊酰半胱氨酸甲基转移酶（Icmt）的a-羧基甲基化是抑制Ras蛋白作用的可选靶点。羧基甲基化是酵母和小鼠细胞中Ras蛋白正确定位的关键。鉴于这一重要发现，我们相信Icmt代表了化疗干预的一个极好的靶点。我们假设某些容易合成的基于异戊二烯类的Icmt抑制剂将成为开发抗ras癌症化疗药物的有价值的先导化合物。我们实验室最近发表的和未发表的研究表明，对戊酰半胱氨酸的氨基修饰和类异戊二烯单元的修饰可以提供小分子的Icmt抑制剂。这项跨学科合作研究计划的具体目标如下：目标1)通过固相合成方案，将新型法尼醇类似物细化为相应的戊酰半胱氨酸衍生物。然后这些将进行进一步的化学修饰，以提供戊酰半胱氨酸衍生物的文库。目的2)这些戊酰半胱氨酸衍生物将作为Icmt的潜在抑制剂进行分析，使用高通量体外分析系统和具有良好特征和强大的Icmt活性单点分析程序。目的3)评估丙烯酰半胱氨酸类似物在体内阻断丙烯酰半胱氨酸甲基化的能力。有效的细胞渗透性抑制剂将评估其在体内错误定位Ras，干扰Ras介导的信号传导，阻断锚定不依赖的胰腺导管癌生长和阻断肿瘤生长的能力。

DESCRIPTION (provided by applicant): Members of the Ras family, implicated in many human cancers, are modified post-translationally, targeting them to the appropriate intracellular location. Ras and other-CaaX proteins undergo 3 sequential reactions: isoprenylation of the cysteine, in particular farnesylation by farnesyltransferase (FTase); proteolysis of the 3 terminal amino acids (-aaX); and a-carboxyl methylation of the isoprenylated cysteine. This process is crucial for membrane localization and thus activity of the key Ras oncoproteins. FTase inhibitors are being evaluated in clinical trials as cancer chemotherapeutic agents. Unfortunately, these compounds have surprisingly little effect on many Ras-transformed tumors. There is now growing interest in the subsequent enzymatic steps; proteolysis by Ras converting enzyme (Reel) and a-carboxyl methylation by isoprenylcysteine methyltransferase (Icmt) as alternative targets for the inhibition of Ras protein action. Carboxyl methylation is critical for the proper localization of Ras proteins in yeast and mouse cells. Given this important finding, we believe that Icmt represents an excellent target for chemotherapeutic intervention. We hypothesize that certain readily synthesized, isoprenoid-based inhibitors of Icmt will be valuable lead compounds for the development of anti-Ras cancer chemotherapeutic agents. Recent published and unpublished studies from our laboratories have demonstrated that modifications to both the amino modification and the isoprenoid unit of prenylcysteines can afford small-molecule inhibitors of Icmt. The specific aims of this collaborative, interdisciplinary research proposal are as follows: Aim 1) Novel farnesol analogs will be elaborated to the corresponding prenylcysteine derivatives, via a solid-phase synthetic protocol. These will then undergo further chemical modification to provide libraries of prenylcysteine derivatives. Aim 2) These prenylcysteine derivatives will be assayed as potential inhibitors of Icmt, using high-throughput in vitro assay systems and well-characterized and robust single point assay procedures for Icmt activity. Aim 3) The prenylcysteine analogues will be evaluated for their ability to block prenylcysteine methylation in vivo. Potent cell permeable inhibitors will be evaluated for their ability to mislocalize Ras, interfere with Ras-mediated signaling, block anchorage-independent growth of pancreatic ductal carcinoma, and block tumor growth in vivo.