Specific Biochemical Inactivation of Oncogenic Ras

描述（由申请人提供）：Ras癌基因是大约三分之一的人类癌症的病原体，目前还没有有效的药物治疗方法专门针对致癌的Ras蛋白。我们在大肠杆菌粗提取物中检测到一种活性，该活性有效地将gtp结合的致癌Ras蛋白转化为无活性的GDP结合形式。对其活性进行了纯化，鉴定出一个负责活性的蛋白为核苷二磷酸激酶（NDK）。我们发现NDK可以特异性灭活几种致癌Ras蛋白，但不能灭活野生型Ras蛋白。我们建议扩展这些发现，以测试体内生物化学灭活致癌Ras作为抑制肿瘤发生手段的可行性。拟建研究的具体目的是：1 .建立NDK促进Ras GTPase致癌物质的特异性失活的生化机制。这有望为了解与致癌Ras蛋白相关的生化缺陷以及促进特异性Ras失活的潜在策略提供见解。2。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的致癌作用的能力。在这里，NDK将被用作研究在体内促进Ras生化失活作为一种治疗策略的可行性的工具。3。为了验证人类肿瘤中NM23 （NDK的人类同源物，先前已被确定为功能未知的肿瘤/转移抑制因子）的选择性缺失与致癌形式Ras的效力增加相关的假设。IV.利用高通量生化筛选策略来识别有可能促进Ras致癌形式失活的小分子。这将与哈佛化学和化学生物学研究所密切合作。总之，拟议的研究有望最终导致一种新的治疗方法的发展，以实现对含有致癌突变形式的Ras的人类肿瘤的特异性靶向。

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.