High-throughput screening for HIV assembly and maturation inhibitors

描述（由申请人提供）：意义：该研究旨在通过开发一种敏感的荧光偏振测定方法来填补抗hiv药物发现方面的重要技术空白，该方法可以自动用于高通量筛选（HTS） HIV-1衣壳蛋白c端结构域二聚化的小分子抑制剂或CCA。由于CCA二聚化对于HIV Gag多蛋白的自我结合和衣壳的形成至关重要，因此这些抑制剂有望消除HIV的组装和成熟，有望成为治疗HIV-1感染的新型治疗剂。背景：fda批准的现有抗hiv药物跨越6个机制类别，靶向逆转录酶、蛋白酶、整合酶、融合酶和进入酶。然而，对所有这些药物类别的耐药性已在体外和体内得到证实。目前迫切需要针对不同病毒成分的新疗法来对抗不断扩大的全球艾滋病毒流行。HIV的组装和成熟虽然是一个重要的抗病毒靶点，但在很大程度上仍未被利用。为了促进靶向HIV组装和成熟的抗病毒药物的发现，还需要开发适合化合物文库HTS的准确、敏感和可重复的检测方法。目的/假设：我们寻求开发一种适用于HTS的荧光偏振测定方法，用于检测HIV小分子抑制剂的组装和成熟。(1)建立并优化HIV衣壳组装小分子抑制剂HTS荧光极化检测方法；(2)筛选fda批准的药物文库，从生物化学和结构上验证阳性命中作为CCA二聚化抑制剂的真实性；(3)验证CCA二聚化抑制剂在原代靶细胞中抑制HIV-1复制的假设。研究设计：我们将用合适的荧光染料对CCA进行定点标记，并使用荧光蛋白作为探针，在文库化合物存在的情况下，使用荧光偏振技术定量CCA二聚体到单体的转变。我们将以384孔的格式对fda批准的药物文库进行初步HTS，并在结构和功能上验证阳性命中，然后在体外测试其抑制成熟样和未成熟样颗粒组装的能力。将测试活性化合物在主要靶细胞中抑制HIV复制的能力。我们的长期目标是开发HIV组装和成熟抑制剂，作为预防HIV-1感染和治疗艾滋病的新型治疗剂。

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.