A Two Hybrid System Based Yeasy Screen for Agents that Affect DNA Damage Checkpoi

描述（由申请人提供）：癌症化疗药物经常引入DNA损伤，因此，触发细胞周期阻滞或凋亡，称为检查点反应。我们已经开发了一种基于双杂交系统的酵母试验，可以检测某些dna损伤治疗后某些检查点蛋白之间增加的相互作用。这种现象伴随着检查点激活，可以通过在特定构建的酿酒酵母菌株的选择性培养基上的菌落生长来检测。有效的癌症化疗通常依赖于药物的组合。该应用程序解决了识别具有抗癌活性的小分子的持续需求，并描述了放大或修改其作用的新型药物的特征。我们计划进一步完善大规模机器人筛选的分析条件，并作为一项原理验证研究，以解决一个复杂的筛选目标：分离出增强（或减弱）既定化疗药物喜树碱检查点激活效应的新化合物。可以预期，这类拓扑异构酶I抑制剂的有用性可以通过提供佐剂来增强其对肿瘤细胞的作用或减弱其对正常细胞的毒性来增强。为此目的，将追求下列具体目标：为了证明将该方法发展成为一种高通量筛选工具的可行性，特别强调筛选修饰喜树碱作用的试剂。2. 修改和测试适当的仪器，以展示高吞吐量的能力。3. 筛选改变喜树碱作用的化合物库。我们计划在第一阶段筛选国家癌症研究所的多样性化合物组（1,900种药物），这些药物已经提供给我们。在这个项目的成功完成后，我们将建立和验证一个独特的、强大的、成本效益高的和快速的筛选系统，并将在分离先导化合物方面取得实质性进展，这些先导化合物可以开发成喜树碱癌症化疗的放大器或佐剂。有效的癌症化疗通常依赖于药物的组合。该应用程序解决了识别具有抗癌活性的小分子的持续需求，并描述了放大或修改其作用的新型药物的特征。北德克萨斯大学健康科学中心（UNTHSC）开发了一种创新技术，可以检测癌症治疗中使用的化疗药物引起的DNA损伤。在这个STTR项目中，UNTHSC将与当地一家高科技公司（UHV Technologies, Inc.）合作，进一步开发这项技术，用于大规模机器人筛选，以分离出增强（或减弱）现有化疗药物喜树碱效果的新化合物。UHV将修改其在先前NIH资助下开发的高通量癌症药物发现系统，通过筛选修饰喜树碱作用的药物化合物库来实施和演示这项新技术。

DESCRIPTION (provided by applicant): Cancer chemotherapy agents frequently introduce DNA damage and, as a consequence, trigger cell cycle arrest or apoptosis, referred to as checkpoint responses. We have developed a two-hybrid-system based yeast assay that detects increased interaction between certain checkpoint proteins following certain DNA-damaging treatments. This phenomenon accompanies checkpoint activation and can be detected by colony growth on selective medium in specially constructed strains of the yeast Saccharomyces cerevisiae. Effective cancer chemotherapy frequently relies on a combination of agents. This application addresses the continued need to identify small molecules that have activity against cancer cells and to characterize novel agents that amplify or modify their effect. We plan to further refine the assay conditions for large-scale robotic screening and, as a proof-of-principle study, to address a sophisticated screening goal: to isolate novel compounds that enhance (or diminish) the checkpoint-activating effect of the established chemotherapy drug camptothecin. It is expected that the usefulness of this agent class of topoisomerase I inhibitors can be enhanced by providing adjuvants that enhance its effect on tumor cells or blunt its toxicity towards normal cells. To this end, the following specific aims will be pursued: 1. To demonstrate the feasibility of developing the assay into a high- throughput screening tool, with special emphasis on screening for agents that modify the camptothecin effect. 2. To modify and test the appropriate instrumentation for demonstration of high throughput capability. 3. To screen a compound library for agents that modify the effects of camptothecin. We plan to screen the National Cancer Institute's Diversity Compound set (1,900 agents) during Phase I that is already available to us. At the successful completion of this project, we will have established and validated a unique, robust, cost efficient and rapid screening system and will have made substantial progress towards isolation of lead compounds that can be developed into amplifiers or adjuvants in camptothecin cancer chemotherapy. Effective cancer chemotherapy frequently relies on a combination of agents. This application addresses the continued need to identify small molecules that have activity against cancer cells and to characterize novel agents that amplify or modify their effect. University of North Texas Health Science Center (UNTHSC) has developed an innovative technique that detects DNA damage caused by chemotherapy agents used in cancer treatment. During this STTR project, UNTHSC will work with a local high technology company (UHV Technologies, Inc.) to further develop this technique for large-scale robotic screening to isolate novel compounds that enhance (or diminish) the effects of the established chemotherapy drug camptothecin. UHV will modify its high throughput cancer drug discovery system developed under previous NIH funding to implement and demonstrate this new technique by screening a compound library for agents that modify the effects of camptothecin.