High-throughput screening for HIV assembly and maturation inhibitors

HIV组装和成熟抑制剂的高通量筛选

基本信息

批准号：
8735972
负责人：
WUYUAN LU
金额：
$ 29.17万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8735972
关键词：
Acquired Immunodeficiency Syndrome Amino Acids Anti-HIV Agents Anti-Retroviral Agents Antiviral Agents BODIPY Biological Assay Biological Availability C-terminal Capsid Capsid Proteins Cells Cessation of life Chemicals Data Analyses Development Dimerization Disease Dose Drug resistance Enzymes Epidemic FDA approved Fluorescence Polarization Fluorescent Dyes Funding Funding Opportunities Gagging HIV HIV Infections HIV drug resistance HIV-1 In Vitro Infection Informatics Institution Integrase Label Lead Libraries Ligation NIH Program Announcements NMR Spectroscopy Patients Peptide Hydrolases Pharmaceutical Preparations Prevention Protein Chemistry Proteins RNA-Directed DNA Polymerase Regimen Research Research Design Resistance Robotics Safety Site Solutions Techniques Testing Therapeutic Therapeutic Agents United States National Institutes of Health Validation Viral Viral Load result Virus Virus Assembly X-Ray Crystallography antiretroviral therapy assay development base combat dimer drug development drug discovery fighting gag Gene Products high throughput screening in vivo inhibitor/antagonist innovation monomer novel particle response screening self assembly small molecule structural biology success synthetic protein therapeutic target tool virology weapons

项目摘要

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.

描述（由申请人提供）：意义：拟议的研究旨在通过开发一种敏感的荧光偏振分析法，以填补抗HIV药物发现中的重要技术空白，该测定可以自动化用于高吞吐量筛选（HTS），以用于高吞吐量筛选（HTS），用于小分子抑制HIV-1 Capsid protein of Capsid Protein of Capsid protein of Capsid protein of Capsid protein of的小分子抑制剂。由于CCA二聚化对于HIV GAG多蛋白和衣壳形成的自我关联至关重要，因此，这种抑制剂有望消除HIV组装和成熟，这有望在HIV-1感染治疗HIV-1。背景：FDA批准的现有抗HIV药物跨越了六个机械类别和靶向逆转录酶，蛋白酶，整合酶，融合和进入。但是，对所有这些药物类别的耐药性已在体外和体内记录。迫切需要针对靶向不同病毒成分的新疗法来打击扩大的全球艾滋病毒流行病。 HIV组装和成熟虽然是一个重要的抗病毒靶标，但基本上仍未开发。为了促进针对HIV组装和成熟的抗病毒药物发现，尚未开发适合复合文库HTS的准确，敏感和可重复的测定法。客观/假设：我们试图开发适用于HTS的荧光极化测定法，用于HIV组装和成熟的小分子抑制剂。具体目的：（1）建立并优化了HTS的HTS荧光极化测定法，用于HIV CAPSID组装的小分子抑制剂；（2）屏幕FDA批准的药物库，并在生化上以及结构上验证阳性命中作为CCA二聚化的真实抑制剂；（3）检验以下假设：CCA二聚化抑制剂抑制原代靶细胞中的HIV-1复制。研究设计：我们将使用合适的荧光染料特定于站点的CCA标记CCA，并使用荧光蛋白作为探针，用于使用荧光极化技术在库化合物的存在下量化CCA二聚体之间的转变。我们将以384孔格式进行FDA批准的药物文库的主要HTS，并在结构和功能上验证正命中率，然后在测试其在体外抑制成熟和不成熟的类似细胞的颗粒的能力之前。活性化合物将测试其抑制原代靶细胞中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，作为一种新型的治疗剂，可预防HIV-1感染和艾滋病治疗。