Inhibition of Prenylated Protein Processing

与许多人类癌症有关的RAS家族成员在翻译后被修饰，靶向于 合适的细胞内位置。RAS和其他-CAAX蛋白经历三个顺序的反应： 半胱氨酸的异戊二烯基化，特别是法尼基转移酶(FTase)的法尼化；三者的蛋白分解 末端氨基酸(-AAX)；和异戊二烯基半胱氨酸的a-羧甲基化。这个过程是 对膜的定位至关重要，从而对关键的RAS癌蛋白的活性至关重要。FTase抑制剂正在被 在临床试验中被评估为癌症化疗药物。不幸的是，这些化合物 令人惊讶的是，对许多RAS转化的肿瘤几乎没有影响。现在越来越多的人对随后的 酶促步骤：RAS转换酶(REEL)的蛋白分解和异戊二烯半胱氨酸的α-羧甲基化 甲基转移酶(ICMT)作为抑制RAS蛋白作用的替代靶点。羧甲基化是 对酵母和小鼠细胞中RAS蛋白的正确定位至关重要。鉴于这一重要发现，我们 认为ICMT是化疗干预的良好靶点。我们假设 某些易于合成的、基于异戊二烯的ICMT抑制剂将是有价值的先导化合物 用于开发抗RAS癌化疗药物。最近出版和未出版 我们实验室的研究表明，对氨基修饰和 戊二烯基半胱氨酸的类异戊二烯单元可以作为ICMT的小分子抑制剂。这样做的具体目的是 协作、跨学科的研究建议如下：目标1)新的法尼醇类似物将是 通过固相合成方案，详细阐述了相应的戊烯基半胱氨酸衍生物。这些遗嘱 然后进行进一步的化学修饰以提供戊烯基半胱氨酸衍生物的文库。目标2)这些 戊烯基半胱氨酸衍生物将成为ICMT的潜在抑制剂，采用高通量体外检测方法。 ICMT活性的系统和特征良好且健壮的单点分析程序。目标3) 将评估戊烯基半胱氨酸类似物在体内阻断戊烯基半胱氨酸甲基化的能力。 有效的细胞通透性抑制剂将被评估其错位定位RAS，干扰RAS- 介导信号转导，阻断胰腺导管癌非锚定生长，阻断肿瘤生长 体内生长。

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 three sequential reactions: isoprenylation of the cysteine, in particular farnesylation by farnesyltransferase (FTase); proteolysis of the three 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 Recent published and unpublished studies from our laboratories have demonstrated that modifications to both the amino modification and the isoprenoid unit ofprenylcysteines can afford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.