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，并且在人类致癌过程中经常失活，主要是通过点突变。一般来说，具有p53突变的癌细胞对目前的放化疗疗法更具抗性。我们的长期目标是发现和开发一类新的小分子药物，通过构象变化将p53从其突变形式中重新激活，作为单独或与放化疗联合作用的抗癌剂。本申请的目的是获得MLPCN（分子库探针生产中心网络）的HTS（高通量筛选），以鉴定和表征诱导突变型p53构象变化的小分子，从而导致其重新激活。我们的中心假设是突变型p53的结构构象可以通过小分子改变为野生型，导致p53的重新激活，从而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筛选300，000种化合物来识别小分子（分子库小分子库），其使用由wt p53响应元件驱动的荧光素酶报告子通过构象变化再活化p53，和2）进行二级确认和反筛选以滤除潜在的假阳性，随后进行p53再活化的生物化学验证和作为单一药剂或与辐射组合的癌细胞杀伤的生物学验证。 筛选试验是在我们的实验室开发的96孔基于细胞的荧光素酶报告基因试验，并已小型化到1536孔板格式。该基于双磷酸酶报告基因的测定使用p53无效的H1299肺癌细胞，该细胞已经用温度敏感的p53突变体p53-A138 V（密码子138处的丙氨酸至缬氨酸突变）和由Mdm 2基因的内含子-1片段驱动的p53响应性BP 100荧光素酶报告基因（命名为H1299-p53 ts-Luc）稳定共转染。将p53构象从突变型改变为野生型的化合物将导致野生型p53依赖性报告物的反式激活，以增加荧光素酶活性。测定窗口可以高达60倍。LOPAC文库的初步筛选鉴定了四种诺考达唑系列化合物，其中三种在反筛选后得到确认。 在成功完成这项拟议的研究，我们希望确定和验证至少一类新的小分子，通过诱导p53突变体的构象变化，重新激活p53。这些分子在SAR（结构活性抑制剂）优化后将具有进一步开发的潜力，用于作为单一药剂以及与放化疗组合用于有效治疗携带突变型p53的人类癌症的临床用途。 公共卫生相关性：肿瘤抑制因子在50%的人类癌症中发生突变，并且具有p53突变的人类癌症通常对化学-放射疗法更具抗性。该提案的目标是鉴定能够恢复p53功能的小分子化合物。这些化合物可能有潜力被进一步开发为单独或与放化疗联合的抗癌药物。

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.