Development of a high throughput HIV assembly screen

描述（由申请人提供）：我们的提案响应了NIH项目公告“高通量药物筛选测定的开发”，其目标是为公共部门研究人员提供采用高通量化学筛选（HTS）方法的机会。该公告要求申请人提出足以显示测定重现性的开发计划;用小型化合物库测试测定;并提供评估HTS命中的重要性的大纲。我们描述了一种独特的检测设计，以确定化合物，抑制艾滋病毒的组装。我们的基于细胞的测定利用了嵌合蛋白可以作为灵敏、高效和可重复的报告基因用于病毒组装的事实，其信噪比应该适合HTS。我们的调查将导致识别的化合物，抑制运输，装配和释放的病毒表达细胞的病毒粒子。这种抑制剂将提供新的HIV抗病毒药物，将有助于解开HIV组装的复杂编排，并可能揭示阻止其他致病病毒复制的方法。因此，我们的研究结果将在了解艾滋病毒的生命周期和阻止病毒感染方面具有普遍适用性。为了达到这些目标，我们的具体目标如下：1。用于高通量筛选的组装测定的优化：将修改组装测定以降低测定间的变异性，改善信噪比，增加筛选系数比，并调整HTS格式的方案。2.使用小化学文库的测定表征：将采用优化的筛选方案来测试不同化合物组的小化学文库。将对试验筛选数据进行评价，以评估统计筛选参数，并将结果用于进一步完善HTS方法。3.制定二级筛选程序：将制定和简化初级筛选结果的分析和优先排序程序。将设计和测试排除伪影和区分受候选抑制剂影响的特定组装步骤的方法。通过这些工作，将实现发现新的HIV抑制剂和阐明新的组装步骤的前景。 公共卫生相关性：我们的调查与公共卫生直接相关。我们建议开发一种高通量筛选，以确定HIV组装的抑制剂。这种抑制剂将成为设计治疗艾滋病新药的先导。

DESCRIPTION (provided by applicant): Our proposal responds to the NIH program announcement "Development of assays for high throughput drug screening," which has the goal of offering public sector researchers opportunities to employ high throughput chemical screening (HTS) methods. The announcement asks applicants to propose development plans sufficient to show assay reproducibility; to test the assay with a small compound library; and to provide an outline for evaluating the significance of HTS hits. We describe a unique assay designed to identify compounds that inhibit HIV assembly. Our cell-based assay takes advantage of the fact that chimeric proteins can serve as sensitive, efficient, and reproducible reporters for virus assembly with signal-to-noise ratios that should be suitable for HTS. Our investigations will lead to the identification of compounds that inhibit transport, assembly and release of virions from virus-expressing cells. Such inhibitors will provide new HIV antivirals, will help unravel the complicated choreography of HIV assembly, and may reveal ways to block the replication of other pathogenic viruses. Thus, the results of our studies will have general applicability in understanding the HIV life cycle and in stopping virus infections. In keeping with program announcement guidelines, to achieve these goals, our specific aims are as follows: 1. Optimization of assembly assays for high throughput screening: Assembly assays will be modified to reduce assay-to-assay variability, improve signal-to-noise ratios, increase screening coefficient ratios, and adapt protocols for HTS formats. 2. Assay characterization using small chemical libraries: Optimized screening protocols will be employed to test small chemical libraries of diverse compound sets. Test screen data will be evaluated to assess statistical screening parameters, and results will be used to further refine methodologies for HTS. 3. Development of secondary screening procedures: Procedures for analysis and prioritization of primary hits will be developed and streamlined. Methods to rule out artifacts, and to discriminate specific assembly steps impacted by candidate inhibitors will be designed and tested. Through these efforts, the prospect of finding novel HIV inhibitors, and elucidating new assembly steps will be realized. Public Health Relevance: Our investigations are directly relevant to public health. We propose to develop a high throughput screen that will identify inhibitors of HIV assembly. Such inhibitors will serve as leads in the design of novel drugs for the treatment of AIDS.