Specific Biochemical Inactivation of Oncogenic Ras

描述（由申请人提供）：Ras致癌基因是约三分之一人类癌症的致病因子，并且没有有效的基于药物的治疗可特异性靶向致癌Ras蛋白。我们检测到一种粗制的E.大肠杆菌提取物，其有效地将GTP结合的致癌Ras蛋白转化为无活性的GDP结合形式。活性被纯化，并且负责活性的单个蛋白被鉴定为核苷二磷酸激酶（NDK）。我们发现，NDK可以特异性地抑制几种致癌Ras蛋白，但不是野生型Ras，在体外和体内。我们建议扩展这些发现，以测试的可行性，生物化学灭活致癌Ras在体内作为抑制肿瘤发生的一种手段。建议研究的具体目标是：I.建立NDK促进Ras GTP酶致癌形式特异性失活的生化机制。这有望为深入了解致癌Ras蛋白相关的生化缺陷和促进特定Ras失活的潜在策略提供帮助。二.在细胞培养转化试验和K-Ras诱导肿瘤的小鼠模型中评估NDK拮抗Ras致癌作用的能力。在这里，NDK将被用作一种工具，用于检查促进Ras在体内的生化失活作为一种治疗策略的可行性。三.检验人肿瘤中NM 23（NDK的人直系同源物，先前已被鉴定为未知功能的肿瘤/转移抑制因子）的选择性丢失与Ras致癌形式的效力增加相关的假设。四.利用高通量生物化学筛选策略来鉴定具有促进Ras致癌形式失活潜力的小分子。这将与哈佛化学和化学生物学研究所密切合作进行。总之，拟议的研究预计将导致最终开发一种新的治疗方法，用于实现特异性靶向人类肿瘤，这些肿瘤含有致癌突变形式的Ras。

DESCRIPTION (provided by applicant): The Ras oncogene is a causative agent in about one-third of all human cancers, and there are no effective drug-based treatments available that specifically target the oncogenic Ras protein. We detected an activity in a crude E. coli extract that efficiently converts GTP-bound oncogenic Ras proteins to an inactive GDP bound form. The activity was purified and a single protein responsible for the activity was identified as nucleoside diphosphate kinase (NDK). We find that NDK can specifically inactivate several oncogenic Ras proteins, but not wild-type Ras, in vitro and in vivo. We propose to extend these findings to test the feasibility of biochemically inactivating oncogenic Ras in vivo as a means of inhibiting tumorigenesis. The Specific Aims of the proposed studies are: I. To establish the biochemical mechanism by which NDK promotes the specific inactivation of oncogenic forms of the Ras GTPase. This is expected to provide insights into the biochemical defect associated with oncogenic Ras proteins and potential strategies for promoting specific Ras inactivation. II. To assess the ability of NDK to antagonize the oncogenic actions of Ras in cell culture transformation assays and in a mouse model of K-Ras-induced tumors. Here, NDK will be utilized as a tool for examining the feasibility of promoting biochemical inactivation of Ras in vivo as a therapeutic strategy. III. To test the hypothesis that selective loss of NM23 (the human ortholog of NDK, which has been identified previously as a tumor/metastasis suppressor of unknown function) in human tumors is associated with increased potency of oncogenic forms of Ras. IV. To utilize a high throughput biochemical screening strategy to identify small molecules with the potential to facilitate inactivation of oncogenic forms of Ras. This will be performed in close collaboration with the Harvard Institute of Chemistry and Chemical Biology. Together, the proposed studies are expected to lead to the eventual development of a novel therapeutic approach for achieving specific targeting of human tumors that harbor oncogenic mutant forms of Ras.