TRACING KINASE CASCADES--SYNTHETIC ATP TAG FOR C ABLAB1

介导磷酸盐从ATP转移到蛋白质的酶， 激酶是最大和最重要的蛋白质家族之一 已知（>250个）。 正是这些特征使激酶成为 几乎每一个致癌和正常的信号传导途径也使特异性 鉴定它们的体内底物极其困难。 我们 在这里提出了一种新的化学方法，直接解决 现有方法的缺陷。 新奇之处在于创造了一个 激酶之间的人工结构差异， 核苷酸辅因子 具体来说，我们建议为C创建一个“标签”- abl酪氨酸激酶，其负调节细胞生长并且是原- 癌基因 c-ab 1染色体易位（费城染色体易位） 染色体）产生融合癌蛋白bcr-abl以及bcr-abl 导致慢性粒细胞白血病（CML）。 我们目前缺乏 关于致癌酪氨酸激酶底物的知识代表了 在发现新的治疗方法中最大的挑战 治疗慢性粒细胞白血病等白血病的策略。 一旦下游 这些癌蛋白的靶点被确定为经典的药物发现 可以应用一些方法来抑制它们的活性并阻断它们的生长。 肿瘤发生 目标#1：设计、合成和测定一组候选化合物 c-ab 1的死底物ATP类似物。 目标#2：生成四个 设计具有新核苷酸特异性的c-ab 1定点突变体 用于独特的合成ATP类似物（F336 A、L389 A、V275 A、L267 A）。 目标#3： 制定一个成功的战略，提供修改非自然[-32P] 三磷酸盐穿过细胞膜。

The enzymes which mediate the transfer of phosphate from ATP to proteins, the kinases, are among the largest and most import families of proteins known (>250 identified). The very features which make kinases a part of almost every oncogenic and normal signaling pathway also renders specific identification of their in vivo substrates extremely difficult. We propose here a novel chemical approach which directly addresses the deficiencies in existing methodologies. The novelty rests in creating an artificial structural distinction between kinases at the level of the nucleotide cofactor. Specifically we propose to create a 'tag' for the C- abl tyrosine kinase which negatively regulates cell growth and is a proto- oncogene. The chromosomal translocation of c-ab1 (the Philadelphia Chromosome) produces the fusion oncoprotein bcr-abl as well as bcr-abl which causes chronic myelogenous leukemia (CML). Our current lack of knowledge about the substrates of oncogenic tyrosine kinases represents the single greatest challenge in the discovery of new therapeutic strategies for fighting may leukemias such as CML. Once downstream targets of these oncoproteins are identified classic drug discovery approaches can be applied to inhibiting their activity and blocking oncogenesis. Aim #1: To design, synthesize, and assay a set of candidate ATP analogs that are dead substrated for c-ab1. Aim #2: To generate four site-directed mutants of c-ab1 designed to have new nucleotide specificity for unique synthetic ATP analogs (F336A, L389A, V275A, L267A). Aim #3: to develop a successful strategy for delivering modified unnatural [-32P] triphosphates across cell membranes.