Development of a high throughput HIV assembly screen

描述（由申请人提供）：我们的提案响应NIH项目公告“开发高通量药物筛选测定法”，其目标是为公共部门研究人员提供使用高通量化学筛选（HTS）方法的机会。该公告要求申请人提出足以证明试验可重复性的开发计划；用一个小的化合物文库进行试验；并为评估高温高温命中的重要性提供一个大纲。我们描述了一种独特的测定方法，旨在识别抑制HIV组装的化合物。我们基于细胞的检测利用了嵌合蛋白可以作为病毒组装的敏感、高效和可重复的报告者这一事实，其信噪比应该适用于HTS。我们的研究将导致鉴定化合物，抑制运输，组装和释放病毒粒子从病毒表达细胞。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性病毒复制的方法。因此，我们的研究结果将在理解HIV生命周期和阻止病毒感染方面具有普遍的适用性。根据节目公告的指导方针，为实现这些目标，我们的具体目标如下：优化高通量筛选的组装分析：组装分析将被修改，以减少分析到分析的可变性，提高信噪比，增加筛选系数比率，并适应HTS格式的协议。2. 使用小化学文库的分析表征：优化的筛选方案将用于测试不同化合物集的小化学文库。将对测试筛选数据进行评估，以评估统计筛选参数，结果将用于进一步完善HTS的方法。3. 制定二次筛选程序：将制定和精简分析和确定主要命中目标优先次序的程序。将设计和测试排除伪影的方法，并区分受候选抑制剂影响的特定组装步骤。通过这些努力，发现新的HIV抑制剂和阐明新的组装步骤的前景将得以实现。

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.