Novel Assays for Inhibitors of HIV Assembly

描述（由申请人提供）：事实证明，抗逆转录病毒药物联合疗法在控制艾滋病毒感染方面非常成功，并显著降低了北美和西欧艾滋病的发病率和死亡率。尽管取得了这一成功，但抑制艾滋病毒复制的新目标是非常需要的，并且将需要新的药物来对付日益严重的抗逆转录病毒耐药性问题。本研究计划的总体目标是开发适合高通量筛选的检测方法，以鉴定抑制HIV组装过程的小分子。鉴定特定的HIV组装抑制剂将有两个主要目的。首先，这些化合物将为在细胞和分子水平上解剖组装过程的不同阶段提供有价值的工具。其次，一些鉴定的化合物可能作为开发新型抗逆转录病毒药物的先导化合物。在这里，我们描述了一个综合的研究计划，旨在开发HIV组装抑制剂的高通量筛选试验。Specific Aim 1的实验将开发并验证以Gag- cfp:Gag- yfp FRET为信号的基于细胞的Gag- Gag相互作用抑制剂筛选试验。该试验将适应HTS能力，并将执行化合物文库的自动筛选。在目标2中，基于fret的检测将得到加强，使用最新的荧光蛋白衍生物和检测技术，产生两种性能更好的第二代检测。一种新的荧光各向异性测定法测量FRET将进行评估。Aim 3的实验将开发并验证我们实验室发现的Gag和AP-3复合物之间新相互作用抑制剂的体外筛选试验。Cy3-Cy5 FRET将是本试验读数的基础。总之，这些检测应该能够鉴定出小分子化合物，这些化合物将为HIV组装领域提供信息，并为进一步解剖组装途径提供重要工具。这些HTS检测也可以应用于筛选通过分子文库计划提供的许多文库，并且旨在满足PA-04- 068，高通量药物筛选检测开发的目标。

DESCRIPTION (provided by applicant): Combination antiretroviral drug regimens have proven remarkably successful at controlling HIV infection and have resulted in significant reductions in morbidity and mortality from AIDS in North America and Western Europe. Despite this success, new targets for inhibition of HIV replication are highly desirable, and new drugs will be needed to combat the rising problem of antiretroviral drug resistance. The overall goal of this research plan is to develop assays suitable for high throughput screening for the identification of small molecules that inhibit the HIV assembly process. The identification of specific HIV assembly inhibitors will serve two major purposes. First, these compounds will provide valuable tools for dissecting distinct stages of the assembly process at the cellular and molecular level. Second, some of the identified compounds may serve as lead compounds for the development of novel antiretroviral drugs. Here we describe an integrated research program designed to develop high-throughput screening assays for inhibitors of HIV assembly. Experiments in Specific Aim 1 will develop and validate a cell-based screening assay for inhibitors of Gag- Gag interactions using Gag-CFP:Gag-YFP FRET as the signal. This assay will be adapted to HTS capability, and automated screening of a compound library will be performed. In Aim 2, the FRET-based assay will be enhanced to produce two second-generation assays with improved performance using the latest fluorescent protein derivatives and assay technologies. A novel fluorescence anisotropy assay for measuring FRET will be evaluated. Experiments in Aim 3 will develop and validate an in-vitro screening assay for inhibitors of a new interaction discovered in our laboratory between Gag and the AP-3 complex. Cy3-Cy5 FRET will be the basis for the readout from this assay. Together, these assays should identify small molecule compounds that will inform the field of HIV assembly and provide important tools for further dissection of assembly pathways. These HTS assays can also be applied to screening of a number of libraries available through the Molecular Libraries Initiative, and are designed to address the goals of PA-04- 068, Development of Assays for High-Throughput Drug Screening.