TRACING KINASE CASCADES--SYNTHETIC ATP TAG FOR C ABLAB1

追踪激酶级联——C ABLAB1 的合成 ATP 标签

• 批准号: 2414450
• 负责人: KEVAN M. SHOKAT
• 金额: $ 15.06万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414450
• 关键词: adenosine triphosphate; biological signal transduction; biomarker; cell growth regulation; chemical synthesis; chromosome translocation; chronic myelogenous leukemia; cofactor; enzyme substrate; enzyme substrate analog; molecular cloning; nucleotide analog; oncoproteins; protein tyrosine kinase; protooncogene; purine analog; radiotracer; site directed mutagenesis

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.

介导磷酸从ATP转化为蛋白质的酶，