Novel Assays for Inhibitors of HIV Assembly

描述（由申请人提供）：抗逆转录病毒药物组合方案已被证明在控制HIV感染方面非常成功，并导致北美和西欧艾滋病发病率和死亡率显著降低。尽管取得了这一成功，但抑制艾滋病毒复制的新目标仍是非常可取的，而且需要新的药物来解决日益严重的抗逆转录病毒药物耐药性问题。本研究计划的总体目标是开发适用于高通量筛选的检测方法，以鉴定抑制HIV组装过程的小分子。识别特定的HIV装配抑制剂将用于两个主要目的。首先，这些化合物将为在细胞和分子水平上剖析组装过程的不同阶段提供有价值的工具。第二，一些已鉴定的化合物可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，旨在开发高通量筛选HIV组装抑制剂的测定。具体目标1中的实验将开发并验证使用Gag-CFP：Gag-YFP FRET作为信号的Gag-Gag相互作用抑制剂的基于细胞的筛选测定。该试验将适应HTS能力，并将对化合物文库进行自动筛选。在目标2中，基于FRET的检测将得到增强，以产生两种第二代检测，使用最新的荧光蛋白衍生物和检测技术提高性能。一种新的荧光各向异性测定测量FRET将进行评估。目标3中的实验将开发和验证在我们实验室中发现的Gag和AP-3复合物之间的新相互作用的抑制剂的体外筛选测定。Cy 3-Cy 5 FRET将是本试验读数的基础。总之，这些检测应该确定小分子化合物，将告知艾滋病毒组装领域，并提供重要的工具，进一步解剖组装途径。这些HTS检测试剂盒也可用于筛选分子库计划提供的大量文库，旨在实现PA-04- 068《高通量药物筛选检测试剂盒的开发》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.