Identifying Small Molecule Inhibitors of HdmX using Cell-based Screening

描述（由申请人提供）：绝大多数人类癌症中发生p53信号传导途径的缺失，无论是通过突变还是上游或下游信号传导组分的缺失。在正常细胞中，Hdm 2和HdmX协调调节p53的稳定性和功能。然而，每种蛋白质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。最近报道，HdmX过表达在乳腺癌、结肠癌和肺癌中高达20%。如果最近发现的能够破坏p53-Hdm 2相互作用以诱导p53依赖性毒性的低分子量化合物继续显示出积极的结果，则携带野生型p53的肿瘤的治疗选择将大大扩展。一种小分子特异性的初步比较表明，HdmX和Hdm 2可以受到不同的影响，从而将每种蛋白质确定为独立的治疗靶点。该提案的目标是开发一类新的小分子，其有效地对抗保留野生型p53的肿瘤细胞，所述野生型p53由于HdmX过表达而被转录抑制。提出了一种基于细胞的系统，该系统将联合收割机HdmX过表达与驱动2-半乳糖苷酶表达的整合的p53应答启动子（一种容易测量和定量的特异性标记）相结合。将进行诱导p53依赖性转录的化合物的筛选。通过初步筛选的候选化合物将根据其相对强度进行分类，并表征以确定p53激活机制和对HdmX和/或Hdm 2状态的依赖性。能够抑制HdmX介导的p53失活的有限的一组化合物将形成II期计划的基础，该计划旨在进行靶向优化和原型治疗药物的开发。公共卫生相关性：癌细胞已经获得了遗传改变，破坏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，从而可以选择重新参与并实施作为癌症治疗的扩散的隐藏限制。我们建议鉴定可以防止具有常见但特异性遗传改变的肿瘤（即HdmX蛋白过度表达）继续其异常生长的化合物。

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.