High-throughput screening for HIV assembly and maturation inhibitors

描述(由申请人提供)：意义：拟议的研究试图通过开发一种灵敏的荧光偏振分析来填补抗艾滋病毒药物开发中的一个重要技术空白，该方法可以自动高通量筛选(HTS)HIV-1衣壳蛋白或CCA的C-末端结构域二聚的小分子抑制剂。由于CCA二聚化对于HIV Gag多聚蛋白的自结合和衣壳的形成至关重要，这类抑制剂有望阻止HIV的组装和成熟，有望成为一类治疗HIV-1感染的新型治疗剂。背景：FDA批准的现有抗HIV药物跨越六个机械类别，靶向为逆转录酶、蛋白酶、整合酶、融合和进入。然而，在体外和体内，已经记录了对所有这些药物类别的耐药性。迫切需要针对不同病毒成分的新疗法来抗击不断扩大的全球艾滋病毒流行。艾滋病毒的组装和成熟，虽然是一个重要的抗病毒目标，但在很大程度上仍未得到开发。为了促进针对HIV组装和成熟的抗病毒药物的发现，适用于化合物文库HTS的准确、灵敏和可重复性的检测方法尚未开发出来。目的/假设：我们试图建立一种适用于HTS的荧光偏振分析方法，以检测HIV组装和成熟的小分子抑制剂。具体目的：(1)建立和优化针对HIV衣壳组装小分子抑制剂的HTS荧光偏振分析；(2)筛选FDA批准的药库，并从生物化学和结构上验证阳性HITS是真正的CCA二聚抑制剂；(3)验证CCA二聚抑制剂抑制HIV-1在原代靶细胞复制的假设。研究设计：我们将用合适的荧光染料定点标记CCA，并使用荧光蛋白作为探针，在库化合物存在的情况下使用荧光偏振技术定量CCA从二聚体到单体的转变。我们将以384孔的形式对FDA批准的药物文库进行初级HTS，并在结构和功能上验证阳性HITS，然后测试它们在体外抑制成熟和未成熟颗粒组装的能力。活性化合物将被测试其抑制HIV在主要目标细胞中复制的能力。我们的长期目标是开发HIV组装和成熟抑制剂，作为一类新型的治疗药物，用于预防HIV-1感染和治疗艾滋病。 公共卫生相关性：目前针对HIV-1感染患者的抗逆转录病毒疗法(ART)使用的是针对病毒酶、逆转录酶和蛋白酶的抑制剂组合。抗逆转录病毒疗法减少了病毒载量，减缓了艾滋病毒向艾滋病的发展，为世界艾滋病死亡人数的稳步下降做出了贡献。尽管ART取得了成功，但它并不能从感染细胞中根除艾滋病毒，而且，在ART的许多并发症中，出现了对目前的抗逆转录病毒疗法无效的抗药性艾滋病毒株。这项拟议的研究旨在开发一种灵敏的荧光偏振分析方法，该方法可以自动高通量筛选HIV-1衣壳蛋白C-末端结构域二聚的小分子抑制剂。这种抑制剂可能会成为武器库中的额外武器，通过专门针对艾滋病毒的组装和成熟来对抗艾滋病毒感染。

DESCRIPTION (provided by applicant): Significance: The proposed research seeks to fill an important technological gap in anti-HIV drug discovery by developing a sensitive fluorescence polarization assay that can be automated for high throughput screening (HTS) for small molecule inhibitors of dimerization of the C-terminal domain of the HIV-1 capsid protein or CCA. Since CCA dimerization is critically important for self-association of the HIV Gag polyprotein and capsid formation, such inhibitors are expected to abrogate HIV assembly and maturation, promising a novel class of therapeutic agents for the treatment of HIV-1 infection. Background: FDA-approved existing anti-HIV drugs span six mechanistic classes and target reverse transcriptase, protease, integrase, fusion, and entry. However, resistance to all these drug classes has been documented in vitro and in vivo. New therapies that target different viral components are urgently needed to combat expanding global HIV epidemics. HIV assembly and maturation, while a significant antiviral target, has remained largely unexploited. To facilitate antiviral drug discovery that targets HIV assembly and maturation, accurate, sensitive and reproducible assays suitable for HTS of compound libraries have yet to be developed. Objective/Hypothesis: We seek to develop a fluorescence polarization assay suitable for HTS for small molecule inhibitors of HIV assembly and maturation. Specific Aims: (1) Establish and optimize a fluorescence polarization assay for HTS for small molecule inhibitors of HIV capsid assembly; (2) Screen FDA-approved drug libraries, and biochemically as well as structurally validate the positive hits as authentic inhibitors of CCA dimerization; (3) Test the hypothesis that CCA dimerization inhibitors suppress HIV-1 replication in primary target cells. Study design: We will site-specifically label CCA with a suitable fluorescent dye, and use the fluorescent protein as a probe for quantifying the CCA dimer-to-monomer transition in the presence of library compounds using fluorescence polarization techniques. We will conduct primary HTS of FDA-approved drug libraries in a 384-well format, and structurally and functionally validate the positive hits before testing their ability to inhibit assembly of both mature-like and immature-like particles in vitro. Active compounds will be tested for their ability to inhibit HIV replication in primary target cells. Our long-term objective is to develop HIV assembly and maturation inhibitors as a novel class of therapeutic agents for the prevention of HIV-1 infection and the treatment of AIDS. PUBLIC HEALTH RELEVANCE: Current antiretroviral therapy (ART) for HIV-1 infected patients utilizes a combination of inhibitors that target the viral enzymes reverse transcriptase and protease. ART reduces viral load and slows the progression of HIV to AIDS, contributing to a steady decrease in AIDS deaths in the world. Despite its success, ART does not eradicate HIV from infected cells, and, among many complications of ART is the emergence of drug-resistant HIV strains not responding to current antiretroviral regimens. The proposed research aims to develop a sensitive fluorescence polarization assay that can be automated for high-throughput screening for small molecule inhibitors of dimerization of the C- terminal domain of the HIV-1 capsid protein. Such inhibitors may become additional weapons in the arsenal to fight HIV infection by specifically targeting HIV assembly and maturation.