High Throughput Screens of Novel Radiation Sensitizers and Protectors

描述（由申请人提供）：这是一项新的申请，汇集了辐射生物学和机器人高通量药物筛选技术的专业知识，以确定用于临床放射肿瘤学和放射恐怖主义事件的新型辐射致敏剂和保护剂。提出了三个具体目标。Specific Aim 1将实施一种高通量的基于细胞的筛选，以鉴定能够使人类细胞对电离辐射敏感/保护细胞免受电离辐射的化合物。这是一个药物发现的目标，将使我们能够从大约40,000种化合物的文库中识别出放射性致敏剂或放射性保护剂的铅分子。特异性Aim 2将通过克隆细胞存活试验验证Aim 1中获得的结果。将在增加辐射剂量的情况下研究不同的药物浓度和照射时间。目标2还将检验辐射防护剂在辐照后使用有效的假设。一旦我们证实了关于致敏或保护的初步观察结果，我们将使用在肿瘤细胞中缺乏已知被破坏基因的一个或两个等位基因的等基因细胞来确定铅化合物在野生型细胞与在癌症中常见的具有特征突变的细胞中是否具有不同的致敏/保护。具体来说，我们将测试一种假设，即与癌细胞中常见的含有遗传改变的细胞相比，化合物对野生型细胞具有不同的敏感性/保护作用。特异性目标3将验证在目标1中确定并在目标2中确认的致敏剂/保护剂不会使存活细胞易受增强基因组不稳定性的影响。在存在致敏剂或保护剂的情况下，暴露于临床相关剂量的辐射不会杀死所有细胞。使用绿色荧光蛋白为基础的报告试验，我们将验证这样的假设，即通过延迟突变和超重组测量，在+/-敏化剂或保护剂照射下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，我们将测试的假设，即后代细胞存活辐照+/-敏化剂或保护剂不太可能表现出基因组不稳定的延迟染色体不稳定性测量。在马里兰大学，所有的技术和细胞系都是可用的。我们有专业知识来确定用于临床和放射性恐怖主义事件的新的辐射致敏剂/保护剂。成功实现拟议的研究目标将对接受放射治疗的患者和涉及放射性物质的恐怖主义事件中的公众作出重大贡献。公共卫生相关性：该应用程序有两个可行且可能非常重要的目标。第一种是利用最先进的技术来鉴定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。第二种是利用同样的技术来发现独特的化合物，可以防止有害的辐射影响，从而在发生放射性事件时保护个人。

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.