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，并因此触发细胞周期停滞或凋亡，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检测方法，该方法可以检测某些DNA损伤处理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，并且可以通过在特别构建的酿酒酵母菌株中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。本申请解决了鉴定对癌细胞具有活性的小分子和表征放大或改变其作用的新型试剂的持续需求。我们计划进一步完善大规模机器人筛选的测定条件，并作为原理验证研究，以解决一个复杂的筛选目标：分离增强（或减弱）已建立的化疗药物喜树碱的检查点激活作用的新型化合物。预期这类拓扑异构酶I抑制剂的有用性可通过提供增强其对肿瘤细胞的作用或减弱其对正常细胞的毒性的佐剂来增强。为此，将追求以下具体目标：1.证明将该测定法开发为高通量筛选工具的可行性，特别强调筛选改变喜树碱效应的药物。2.修改和测试适当的仪器，以证明高通量能力。3.从化合物库中筛选喜树碱修饰剂。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集（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，我们将建立和验证一个独特的，强大的，具有成本效益和快速筛选系统，并将取得重大进展，可开发成喜树碱癌症化疗放大器或佐剂的先导化合物的分离。有效的癌症化疗通常依赖于药物的组合。本申请解决了鉴定对癌细胞具有活性的小分子和表征放大或改变其作用的新型试剂的持续需求。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。在这一短期技术研究所项目期间，联合国人类住区中心将与当地一家高科技公司（特高压技术公司）合作。进一步开发这种技术，用于大规模的机器人筛选，以分离出增强（或减弱）已建立的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，以通过筛选化合物库来实施和展示这种新技术，以改变喜树碱的作用。

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.