Luciferase-based screening for p53 conformational activators

描述(申请人提供)：肿瘤抑制因子在抑制肿瘤发生中起关键作用，在人类癌变过程中经常失活，主要是通过点突变。一般来说，带有p53突变的癌细胞对当前的放化疗疗法更具抵抗力。我们的长期目标是发现和开发一类新的小分子药物，通过构象变化重新激活突变形式的p53，作为单独作用或与化疗联合作用的抗癌药物。该应用程序的目标是访问MLPCN(分子文库探测生产中心网络)的HTS(高通量筛选)，以识别和表征导致突变型p53构象变化并导致其重新激活的小分子。我们的中心假设是突变型P53的结构构象可以被一个小分子改变为野生型，导致P53的重新激活，通过诱导生长停滞和凋亡来抑制癌细胞的生长。具体目标是1)通过在MLSMR(分子文库小分子储存库)筛选300,000种化合物来识别小分子，这些小分子使用由wt P53响应元件驱动的荧光素酶报告来通过构象变化重新激活P53；2)进行二次确认和反筛选以过滤掉潜在的假阳性，随后是对P53重新激活的生化验证以及对作为单一药物或与放射联合应用杀死癌细胞的生物验证。该筛选试验是在我们实验室以96孔细胞为基础的荧光素酶报告试验中发展起来的，并已被微型化为1536孔板格式。这种基于荧光素酶报告基因的分析方法使用了P53缺失的H1299肺癌细胞，这些细胞已经稳定地与温度敏感型P53突变体、P53-A138V(第138密码子上的氨基酸到Valine的突变)和由MDM2基因内含子-1片段(标记为H1299-p53ts-Luc)驱动的P53响应的BP100-荧光素酶报告基因共转染。将p53构象从突变型改变为野生型的化合物将导致依赖于p53的报告的反式激活，从而提高荧光素酶的活性。检测窗口可以高达60倍。对Lopac文库的初步筛选确定了四个诺可达唑系列化合物，其中三个在反筛选后得到确认。在这项拟议的研究成功完成后，我们预计将鉴定和验证至少一类新的小分子，它们通过诱导p53突变体的构象变化来重新激活p53。经过结构活性关系(SAR)优化后，这些分子将具有进一步开发的潜力，可作为单一药物用于临床，以及与化疗放射联合用于有效治疗携带突变型p53的人类癌症。 公共卫生相关性：肿瘤抑制基因在50%的人类癌症中发生突变，具有p53突变的人类癌症通常对化疗放射治疗更具抵抗力。这项提议的目标是鉴定能够恢复P53功能的小分子化合物。这些化合物有可能单独或与化学放射联合作为抗癌药物进一步开发。

DESCRIPTION (provided by applicant): Tumor suppressor plays a key role in suppression of tumorigenesis and is frequently inactivated, mainly by point mutation, during human carcinogenesis. In general, cancer cells with a p53 mutation are more resistant to current chemoradiation therapies. Our long range goal is to discover and develop a novel class of small molecule drugs, that reactivate p53 from its mutant form via conformational changes, as anticancer agents acting alone or in combination with chemoradiation. The objective of this application is to gain access to the HTS (High Throughput Screening) at the MLPCN (Molecular Libraries Probe Production Centers Network) to identify and characterize small molecules that induce conformational changes in mutant p53, leading to its reactivation. Our central hypothesis is that structural conformation of a mutant p53 can be changed to wild type by a small molecule, leading to reactivation of p53 to suppress growth of cancer cells by inducing growth arrest and apoptosis. Specific aims are 1) to identify small molecules, via screening 300,000 compounds at the MLSMR (Molecular Libraries Small Molecule Repository), that reactivate p53 through conformational change using a luciferase reporter driven by a wt p53 responsive element and 2) to conduct secondary confirmation and counterscreening to filter out potential false positives, followed by biochemical validation for p53 reactivation and biological validation for cancer cell killing as single agent or in combination with radiation. The screening assay was developed in our laboratory in a 96-well cell-based luciferase reporter assay and has been miniaturized into a 1536-well plate format. This luciferase-reporter-based assay uses p53-null H1299 lung cancer cells which have been stably co-transfected with a temperature sensitive p53 mutant, p53-A138V (analine-to-valine mutation at codon 138), and a p53-responsive BP100-luciferase reporter, driven by intron-1 fragment of the Mdm2 gene (designated as H1299-p53ts-Luc). The compound that changes p53 conformation from mutant to wild type will result in transactivation of a wt p53-dependent reporter for increased luciferase activity. The assay window can be as high as 60-fold. Initial screening of the LOPAC library identified four nocodazole series of compounds and three were confirmed after a counterscreening. Upon successful completion of this proposed study, we expect to identify and validate at least one novel class of small molecules that reactivate p53 via inducing conformational change of p53 mutants. These molecules would have the potential to be further developed, after SAR (Struction Activity Relationsip) optimization, for clinic use as a single agent as well as in combination with chemoradiation for effective treatment of human cancer harboring a mutant p53. PUBLIC HEALTH RELEVANCE: Tumor suppressor is mutated in 50% human cancers and human cancers with p53 mutation are in general more resistant to chemo-radiation therapy. The goal of this proposal is to identify small molecule compounds that are able to restore p53 function. These compounds would likely have the potential to be further developed as anticancer agents alone or in combination with chemoradiation.