High throughput screen for inhibitors of the mdm2/mdmx interaction

描述（由申请人提供）：癌症是美国第二大死亡原因：每4例死亡中就有1例归因于癌症。肿瘤样品的遗传和分子谱分析已经导致可以被小分子靶向以获得治疗益处的蛋白质的鉴定。一个这样的例子是p53肿瘤抑制途径。p53基因仍然是野生型的，但在大约50%的人类癌症中，该蛋白质在功能上是失活的。在这些情况下，经常观察到mdm 2和mdmx（p53的主要负调节因子）的过度表达。Mdm 2是E3泛素连接酶，催化泛素从E2泛素缀合酶转移到p53。这反过来又导致p53的蛋白酶体依赖性降解。许多药物发现工作都集中在破坏mdm 2/p53相互作用，以增加肿瘤中的p53活性。然而，目前mdm 2特异性拮抗剂不能破坏mdmx与p53的相互作用。此外，最近的数据表明，mdm 2与mdmx的相互作用，通过所谓的RING结构域，产生最有效的E3连接酶活性。因此，鉴定能够抑制mdm 2/mdmx相互作用的小分子可能为癌症中p53的稳定和激活提供新的方法。本提案的具体目的描述了MLCPN小分子文库的基于细胞的筛选，以发现mdm 2/mdmx RING-RING相互作用的小分子抑制剂。另外的后续研究，将验证特异性的“命中”，其疗效在肿瘤细胞表达野生型p53。这些研究的成功完成将提供小分子可以抑制mdm 2/mdmx相互作用的原理证明，从而导致p53的稳定和激活。在该筛选中的主要命中物的化学修饰将产生用于基础科研的有价值的化学探针，以及可用于药物开发的潜在先导化合物。 公共卫生相关性：在美国，癌症在死亡原因中仅次于心血管疾病，是世界范围内的头号杀手。p53肿瘤抑制因子是野生型的，但在所有癌症中约50%的癌症中功能失活，并且是小分子抗癌药物重新激活的有吸引力的靶点。该提案的目的描述了一种鉴定新型p53激活化合物的方法，其成功完成将对未来治疗携带野生型p53的癌症产生影响。

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.