Development of a high throughput HIV assembly screen

描述(由申请人提供)：我们的提案响应了美国国立卫生研究院高通量药物筛选检测方法的项目公告，该项目的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。该公告要求申请者提出足够显示化验重复性的开发计划；用一个小的化合物文库测试化验；并提供评估HTS命中重要性的大纲。我们描述了一种独特的实验，旨在识别抑制HIV组装的化合物。我们基于细胞的分析利用了这样一个事实，即嵌合蛋白可以作为敏感、高效和可重现的病毒组装报告，其信噪比应该适合HTS。我们的研究将导致识别抑制病毒表达细胞中病毒粒子的运输、组装和释放的化合物。这些抑制剂将提供新的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理解艾滋病毒生命周期和阻止病毒感染方面具有普遍适用性。为了与计划宣布指南保持一致，为了实现这些目标，我们的具体目标如下：1.优化用于高通量筛选的组装分析：组装分析将进行修改，以降低分析与分析的可变性，提高信噪比，增加筛选系数比率，并调整适合HTS格式的方案。2.使用小化学库的分析表征：将使用优化的筛选方案来测试不同化合物集的小化学库。将对测试筛选数据进行评估，以评估统计筛选参数，结果将用于进一步改进高温超导的方法。3.制定二次筛选程序：将制定和简化一次打击的分析和轻重缓急程序。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。通过这些努力，寻找新的HIV抑制剂和阐明新的组装步骤的前景将被实现。 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，以识别HIV组装的抑制物。这些抑制剂将成为设计治疗艾滋病的新药的先导。

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.