High throughput screen for inhibitors of the mdm2/mdmx interaction

描述(申请人提供)：癌症是美国第二大死因：每4例死亡中就有1例可归因于这种疾病。对肿瘤样本的遗传和分子图谱分析导致了可能被小分子靶向治疗的蛋白质的识别。一个这样的例子是P53肿瘤抑制通路。P53基因仍然是野生型，但在大约50%的人类癌症中，该蛋白处于功能失活状态。在这些病例中，经常可以观察到p53的主要负调控因子MDM2和MDMX的过度表达。MDM2是一种E3泛素连接酶，催化泛素从E2泛素结合酶(S)转移到P53。这进而导致蛋白酶体依赖的P53的降解。许多药物发现工作都集中在破坏MDM2/P53的相互作用，以增加肿瘤中P53的活性。然而，目前针对MDM2的拮抗剂不能破坏MDMX与p53的相互作用。此外，最近的数据表明，MDM2与MDMX通过所谓的环区相互作用，产生最有效的E3连接酶活性。因此，识别能够抑制MDM2/MDMX相互作用的小分子可能为肿瘤中p53的稳定和激活提供新的途径。该提案的具体目标描述了一种基于细胞的MLCPN小分子文库筛选，以发现MDM2/MDMX环环相互作用的小分子抑制剂。其他后续研究将验证‘HITS’的特异性，以及它们在表达野生型p53的肿瘤细胞中的有效性。这些研究的成功完成将提供小分子可以抑制MDM2/MDMX相互作用的原理证明，导致P53的稳定和激活。此屏幕中的初级Hit的化学修饰将产生用于基础科学研究的有价值的化学探针，以及可能被用于药物开发的潜在先导化合物。 公共卫生相关性：在美国，癌症是仅次于心血管疾病的第二大死因，也是全球头号杀手。P53抑癌基因是野生型，但在约50%的癌症中功能失活，是小分子抗癌药物重新激活的有吸引力的靶点。这项提案的目的是描述一种识别一类新的p53激活化合物的方法，它们的成功完成将对未来携带野生型p53的癌症的治疗产生影响。

DESCRIPTION (provided by applicant): Cancer is the second leading cause of death in the United States: 1 in every 4 deaths is attributable to the disease. Genetic and molecular profiling of tumor samples has lead to the identification of proteins that may be targeted by small molecules for therapeutic benefit. One such example is the p53 tumor suppressor pathway. The p53 gene remains wild type, yet the protein is functionally inactivated in approximately 50% of all human cancers. In these cases, overexpression of mdm2 and mdmx, the major negative regulators of p53, is frequently observed. Mdm2 is an E3 ubiquitin ligase, and catalyzes the transfer of ubiquitin from E2 ubiquitin conjugating enzyme(s) to p53. This in turn leads to proteasome-dependent degradation of p53. Many drug discovery efforts have focused on disruption of the mdm2/p53 interaction in order to increase p53 activity in tumors. However, current mdm2-specific antagonists cannot disrupt the interaction of mdmx with p53. Furthermore, recent data indicate that the interaction of mdm2 with mdmx, via so-called RING domains, engenders the most effective E3 ligase activity. Therefore, the identification of small molecules that can inhibit the mdm2/mdmx interaction may provide a new approach to p53 stabilization and activation in cancer. The Specific Aims of this proposal describe a cell-based screen of the MLCPN small molecule library to discover small molecule inhibitors of the mdm2/mdmx RING-RING interaction. Additional follow-up studies are presented that will validate the specificity of 'hits', and their efficacy in tumor cells that express wild type p53. The successful completion of these studies would provide proof-of-principle that small molecules can inhibit the mdm2/mdmx interaction, leading to stabilization and activation of p53. The chemical modification of primary hits in this screen will generate valuable chemical probes for basic scientific research, as well as potential lead compounds that may be taken forward for drug development. PUBLIC HEALTH RELEVANCE: Cancer ranks second to cardiovascular diseases in causes of death in the United States, and is a leading killer worldwide. The p53 tumor suppressor is wild type but functionally inactivated in ~50% of all cancers, and is an attractive target for reactivation by small molecule anticancer drugs. The Aims of this proposal describe an approach to identify a novel class of p53 activating compounds, and their successful completion will have implications for future treatment of cancers harboring wild type p53.