High Throughput Screens of Novel Radiation Sensitizers and Protectors

描述(由申请人提供)：这是一项新的应用程序，它结合了辐射生物学和机器人高通量药物筛选技术的专业知识，以确定新的辐射敏感剂和保护剂，用于临床放射肿瘤学和在发生放射恐怖主义时使用。提出了三个具体目标。具体目标1将实施高通量基于细胞的筛选，以确定能够使人类细胞对电离辐射敏感/保护其免受电离辐射影响的化合物。这是一个药物发现的目标，它将使我们能够从一个由大约40,000种化合物组成的库中识别出具有辐射敏感剂或辐射保护剂的铅分子。特定的AIM 2将使用克隆细胞存活分析来验证在AIM 1中获得的结果。随着辐射剂量的增加，将调查不同的药物浓度和暴露时间。AIM 2还将检验辐射防护器在辐射后使用时有效的假设。一旦我们确认了关于致敏或保护的初步观察，我们将使用缺乏已知在肿瘤细胞中被破坏的基因的一个或两个等位基因的同基因细胞来确定先导化合物是否在野生型细胞中与在癌症中常见的具有良好特征的突变的细胞中具有不同的增敏/保护作用。具体地说，我们将测试这一假设，即与含有通常在癌细胞中发现的基因改变的细胞相比，化合物将不同地敏化/保护野生型细胞。特定的目标3将检验这样的假设，即在目标1中确定并在目标2中确认的敏感剂/保护剂不会使存活的细胞易于增加基因组的不稳定性。在致敏剂或保护剂存在的情况下，暴露于临床相关剂量的辐射不会杀死所有细胞。使用基于绿色荧光蛋白的报告分析，我们将检验这一假设，即通过延迟突变和超重组来测量，在+/-增敏剂或保护剂中存活的细胞的后代不太可能表现出基因组不稳定性。使用微核试验，我们将检验这样一种假设，即在+/-增敏剂或保护剂照射后存活的后代细胞不太可能表现出延迟的染色体不稳定性。马里兰大学的所有技术和细胞系都是可用的，并且已经到位。我们有专门知识来确定新的辐射敏感剂/保护剂，以便在临床和发生放射性恐怖主义时使用。成功实现拟议的研究目标将是对接受放射治疗的患者的重大贡献，也是在涉及放射性材料的恐怖主义事件中对公众的重大贡献。公共卫生承诺：这项应用有两个可行且可能非常重要的目标。首先是使用最先进的技术来识别能够使癌细胞对辐射诱导的细胞杀伤敏感的化合物，从而改善放射治疗。第二是利用这些相同的技术来寻找独特的化合物，这些化合物可以防止有害的辐射影响，从而在发生辐射事故时保护个人。

DESCRIPTION (provided by applicant): This is a new application that brings together expertise in radiation biology and robotic high throughput drug screen technology to identify new radiation sensitizers and protectors for use in clinical radiation oncology and in the event of radiological terrorism. Three specific aims are proposed. Specific Aim 1 will implement a high throughput cell-based screen to identify compounds that can sensitize/protect human cells to/from ionizing radiation. This is a drug discovery aim that will allow us to identify lead molecules that are radio-sensitizers or radio-protectors from a library consisting of ~40,000 compounds. Specific Aim 2 will validate the results obtained in Aim 1 using clonogenic cell survival assays. Different drug concentrations and exposure times will be investigated at increasing radiation doses. Aim 2 will also test the hypothesis that radiation protectors are effective when used after irradiation. Once we have confirmed the initial observations regarding sensitization or protection, we will use isogenic cells that lack one or both alleles of genes known to be disrupted in tumor cells to determine whether lead compounds sensitize/protect differentially in wild type cells versus cells with well characterized mutations commonly found in cancer. Specifically we will test the hypothesis that compounds will differentially sensitize/protect wild type cells compared with cells containing genetic alterations commonly found in cancer cells. Specific Aim 3 will test the hypothesis that the sensitizers/protectors identified in Aim 1 and confirmed in Aim 2 will not predispose surviving cells to enhanced genomic instability. Exposure to clinically relevant doses of radiation in the presence of sensitizers or protectors will not kill all cells. Using a green fluorescence protein-based reporter assay we will test the hypothesis that the progeny of cells surviving irradiation +/-sensitizer or protector are not more likely to manifest genomic instability as measured by delayed mutation and hyperrecombination. Using a micronucleus assay we will test the hypothesis that the progeny cells surviving irradiation +/- sensitizer or protector are not more likely to manifest genomic instability as measured by delayed chromosomal instability. All technology and cell lines are available and in place at the University of Maryland. We have the expertise to identify new radiation sensitizers/protectors for use in the clinic and in the event of radiological terrorism. Successfully achieving the proposed research goals will be a significant contribution to patients undergoing radiation therapy and to the public in the event of terrorism involving radioactive material. PUBLIC HEALTH RELEVENCE: There are two feasible and potentially very significant goals to this application. The first is to use state of the art technologies to identify compounds that can sensitize cancer cells to radiation- induced cell killing and thus improve radiation therapy. The second is to use those same technologies to find unique compounds that can protect against deleterious radiation effects and thus protect individuals in the event of a radiological incident.