Identifying Small Molecule Inhibitors of HdmX using Cell-based Screening

描述(申请人提供)：P53信号通路的丢失，无论是由于突变或上游或下游信号成分的丢失，在绝大多数人类癌症中都会发生。在正常细胞中，Hdm2和HdmX协同调节P53的稳定性和功能。然而，每种蛋白在许多不同类型的人类恶性肿瘤的亚群中过表达，导致野生型p53功能失活。最近有报道称，HdmX在乳腺癌、结肠癌和肺癌中的过度表达高达20%。如果最近发现的能够破坏P53-Hdm2相互作用从而诱导P53依赖毒性的低分子化合物继续显示出积极的结果，那么含有野生型P53的肿瘤的治疗选择将大大扩大。对一个小分子的特异性的初步比较表明，HdmX和Hdm2可以受到不同的影响，从而将每种蛋白质确立为一个独立的治疗靶点。这项提议的目标是开发一类新的小分子，有效地对抗肿瘤细胞，保留由于HdmX过表达而转录抑制的野生型p53。提出了一种基于细胞的系统，该系统将HdmX的过度表达与整合的P53反应启动子结合起来，驱动2-半乳糖苷酶的表达，这是一种易于测量和量化的特异性标记。将对诱导p53依赖转录的化合物进行筛选。通过初级筛选的候选化合物将根据它们的相对强度进行分类并进行表征，以确定P53激活的机制以及对HdmX和/或Hdm2状态的疗效的依赖。一组有限的能够抑制HdmX介导的P53失活的化合物将形成旨在Hit-to-Lead优化和原型治疗药物开发的第二阶段计划的基础。与公共卫生相关：癌细胞已经获得了基因改变，这些改变扰乱了为限制正常增殖而建立的细胞检查点。在许多癌症中，这些检查点只是被抑制而不是不存在，留下了重新参与并强制实施隐藏的增殖限制作为癌症治疗的选择。我们建议确定能够阻止具有常见但特异的基因改变的肿瘤的化合物，即HdmX蛋白过度表达，以防止其异常生长。

DESCRIPTION (provided by applicant): Loss of the p53 signaling pathway, either by mutation or loss of upstream or downstream signaling components, occurs in the vast majority of human cancers. In normal cells, Hdm2 and HdmX coordinately regulate the stability and function of p53. However, each protein is overexpressed in subsets of many different types of human malignancy, leading to inactivation of wild-type p53 function. HdmX overexpression was recently reported to be as high as 20% in breast, colon and lung cancers. Treatment options for tumors harboring wild-type p53 would be greatly expanded if the recent discoveries of low molecular weight compounds capable of disrupting the p53-Hdm2 interaction to induce p53-dependent toxicity continue to show positive results. Preliminary comparison of one small molecule's specificity has shown that HdmX and Hdm2 can be differentially affected, thereby establishing each protein as an independent target for therapy. The goal of this proposal is to develop a new class of small molecules effective against tumor cells retaining wild-type p53 that is transcriptionally repressed due to HdmX overexpression. A cell-based system is proposed that will combine HdmX overexpression with an integrated p53-responsive promoter driving 2- galactosidase expression, an easily measurable and quantifiable specific marker. A screen for compounds that induce p53-dependent transcription will be performed. Candidate compounds that pass through the primary screen will be classified according to their relative strengths and characterized to define the mechanism of p53 activation and the dependence upon HdmX and/or Hdm2 status for efficacy. A limited set of compounds capable of inhibiting HdmX-mediated p53 inactivation will form the basis of a Phase II program aimed at hit-to-lead optimization and the development of prototype therapeutic drugs. PUBLIC HEALTH RELEVANCE: Cancer cells have acquired genetic alterations that disrupt cellular checkpoints established to limit normal proliferation. In many cancers, these checkpoints are simply suppressed rather than absent, leaving the option to reengage and enforce the hidden limits to proliferation as cancer therapy. We propose to identify compounds that can prevent tumors with a common but specific genetic alteration, namely HdmX protein overexpression, from continuing their aberrant growth.