Luciferase-based screening for p53 conformational activators

描述（由申请人提供）：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，p53突变的癌细胞对目前的放化疗更有抵抗力。我们的长期目标是发现和开发一种新型的小分子药物，通过构象变化使p53从突变形式中重新激活，作为抗癌药物单独作用或与放化疗联合作用。该应用程序的目的是获得MLPCN（分子文库探针生产中心网络）的HTS（高通量筛选），以识别和表征诱导突变型p53构象变化的小分子，从而导致其再激活。我们的中心假设是，突变型p53的结构构象可以通过一个小分子改变为野生型，导致p53的再激活，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（分子文库小分子库）筛选300,000种化合物，识别小分子，这些化合物使用由wt p53响应元件驱动的荧光素酶报告基因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。随后进行了p53再激活的生化验证和单用或联合放疗杀死癌细胞的生物学验证。筛选试验是在我们实验室开发的96孔细胞荧光素酶报告试验，并已小型化为1536孔板格式。这种基于荧光素酶报告基因的检测使用p53缺失的H1299肺癌细胞，这些细胞已经稳定地共转染了温度敏感的p53突变体p53- a138v（密码子138上的analine-to-valine突变）和p53应答的bp100 -荧光素酶报告细胞，由Mdm2基因的内含子-1片段（指定为H1299-p53ts- luc）驱动。将p53构象从突变型改变为野生型的化合物将导致wt p53依赖性报告基因的反激活，从而增加荧光素酶活性。分析窗口可高达60倍。LOPAC文库的初步筛选鉴定出4个nocodazole系列化合物，其中3个经反筛选得到确认。在这项研究的成功完成后，我们希望鉴定和验证至少一类新的小分子，这些小分子通过诱导p53突变体的构象变化来重新激活p53。经过SAR（结构活性关系）优化后，这些分子将有进一步开发的潜力，可作为临床单药使用，也可与放化疗联合使用，有效治疗含有突变p53的人类癌症。

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.