High-Throughput Screening for Multifunctional Nef Inhibitors: Targeting HIV through Revitalizing Immune Defense Mechanisms

多功能 Nef 抑制剂的高通量筛选：通过振兴免疫防御机制靶向 HIV

• 批准号: 10116282
• 负责人: John C. Guatelli
• 金额: $ 22.56万
• 依托单位: UNIVERSITY OF MASSACHUSETTS DARTMOUTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116282
• 关键词: Acquired Immunodeficiency Syndrome; Adopted; Affinity; Anti-HIV Agents; Anti-Retroviral Agents; Antigen Presentation; Area; Attention; Binding; Binding Sites; Biochemical; Biological; Biological Assay; CD4 Positive T Lymphocytes; Cell surface; Cells; Clathrin; Clathrin Adaptors; Collaborations; Complex; Crystallization; Cytoplasmic Tail; Cytotoxic T-Lymphocytes; Defense Mechanisms; Dependence; Detection; Development; Disease; Disease Progression; Dose; Down-Regulation; Drug Evaluation; Drug Screening; Ensure; Epitopes; Fluorescence; Fluorescence Polarization; Goals; HIV; HIV Infections; HIV-1; Human; Immune; Immune system; Immunity; Immunologic Surveillance; In Vitro; Infection; Knowledge; Laboratories; Lead; Major Histocompatibility Complex; Measures; Mediating; Methods; Molecular Conformation; Natural Killer Cells; Pathogenesis; Patients; Pharmaceutical Preparations; Proteins; Resolution; Site; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic; Transcription Factor AP-1; Validation; Viral; Viral Antigens; Virus; Virus Diseases; Virus Replication; Work; antibody detection; antibody-dependent cell cytotoxicity; base; combat; design; drug candidate; drug discovery; enhancer-binding protein AP-2; env Gene Products; experience; experimental study; fighting; high throughput screening; humanized mouse; inhibitor/antagonist; innovation; interest; mouse model; nef Genes; nef Protein; novel; pre-clinical; protein complex; recruit; screening; small molecule libraries; success

PROJECT SUMMARY: Current antiretrovirals control HIV infections but do not cure the disease. An underdeveloped area in anti-HIV drug discovery is to revitalize and harness the power of the immune system to combat and even eliminate HIV. The HIV-1 Nef protein modulates host immunity to promote disease progression to AIDS. Two of the most sig- nificant functions of Nef are surface downregulation of CD4 and surface downregulation of class I major histo- compatibility complex (MHC-I). Nef’s action on CD4 ensures proper processing of an important component of the virus, the viral Env protein. This action also helps the virus conceal epitopes and escape detection by anti- bodies, thereby avoiding antibody-dependent cellular cytotoxicity (ADCC) that is mediated by the host’s natural killer cells for clearing the infection. In addition, Nef disrupts another essential immune recognition mechanism by downregulating MHC-I. Nef’s action here blocks antigen presentation, which is mediated by MHC-I at the surface of the infected cells. Consequently, due to the lack of viral antigen on the cell surface, infections are hidden from immune surveillance and thus successfully evade the killing by the cytotoxic T cells. Given Nef’s abilities to block these key immune mechanisms, it is conceivable that inhibitors of Nef may reverse such ma- nipulation, which should enable detection and possibly clearance of the infection by the host immune system. Convinced by this hypothesis, we aim to develop antiretrovirals through Nef inhibition, which is our long-term goal. In this project, we are inspired by our recent structural findings that Nef uses a common binding site to execute both functions described above. This is exciting because we could potentially use a single drug to block both essential functions, which may restore tremendous immune power to fight and even eliminate HIV. We propose to use a paralleled approach to identify inhibitors capable of binding to this multifunctional site on Nef. Uniquely, we have designed our drug screening experiments in a way that Nef can adopt biologically ac- tive conformations due to the presence of its coopted cellular partners, namely clathrin AP complexes. Our ap- proach is innovative because our design ensures proper formation of the substrate-binding pockets on Nef that involve the targeted multifunctional site. Our specific aims are: 1) identify inhibitors that block CD4 downregula- tion by Nef. Here we will use both an in vitro fluorescence polarization-based, high throughput screening (HTS) assay and a cell-based CD4 downregulation assay to search for Nef inhibitors that block CD4 binding and downregulation; 2) identify inhibitors that block MHC-I downregulation by Nef. Here we will use a similar fluo- rescence polarization-based HTS assay for screening inhibitors of Nef that can disrupt the binding of MHC-ICD to Nef and clathrin AP1. Successful completion of this work will yield dual-functional inhibitors of Nef, which may serve as drug candidates to be further developed into novel, real world antiretrovirals with unparalleled therapeutic potentials.

项目摘要： 当前的抗逆转录病毒控制HIV感染，但不能治愈该疾病。反HIV的欠发达地区 药物发现是为了振兴和利用免疫系统对抗甚至消除艾滋病毒的力量。 HIV-1 NEF蛋白调节宿主免疫以促进疾病向艾滋病的进展。最sig- NEF的重要功能是CD4的表面下调和I类主要组织的表面下调 兼容复合物（MHC-I）。 NEF对CD4的行动可确保正确处理的重要组成部分 病毒，病毒env蛋白。此作用还有助于病毒掩盖表位并通过抗逃脱检测 身体，从而避免由宿主自然介导的抗体依赖性细胞毒性（ADCC） 杀手细胞清除感染。此外，NEF破坏了另一种基本的免疫识别机制 通过下调MHC-I。 NEF的动作在这里阻止了抗原表现，该抗原介导了MHC-I在 感染细胞的表面。因此，由于细胞表面缺乏病毒抗原，感染是 隐藏在免疫监测中，从而成功地逃避了细胞毒性T细胞的杀戮。给定nef 能够阻止这些关键免疫机制的能力，可以想象，NEF的抑制剂可能会扭转这种MA- 牙齿，应能够通过宿主免疫系统检测并可能清除感染。 通过这一假设，我们旨在通过NEF抑制来发展抗逆转录病毒，这是我们的长期 目标。在这个项目中，我们受到了最近的结构发现的启发，即NEF使用一个共同的绑定位点 执行上述两个功能。这很令人兴奋，因为我们可能会使用一种药物来 阻止这两个基本功能，这可能会恢复战斗甚至消除艾滋病毒的巨大免疫力。 我们建议使用并行的方法来识别能够与该多功能位点结合的抑制剂 nef。独特的是，我们以NEF的方式设计了我们的药物筛查实验 由于其持有的细胞伴侣的存在，即clathrin ap复合物，因此具有构象。我们的ap- PRACH具有创新性，因为我们的设计确保了NEF上的基材结合口袋的正确形成 涉及目标多功能站点。我们的具体目的是：1）确定阻止CD4倒立的抑制剂 - nef。在这里，我们将使用基于体外荧光极化高吞吐量筛选（HTS） 测定和基于细胞的CD4下调测定法，以搜索阻断CD4结合和的NEF抑制剂 下调； 2）确定通过NEF阻断MHC-I下调的抑制剂。在这里，我们将使用类似的fluo- 基于恢复偏振的HTS分析，用于筛选NEF的抑制剂，可能破坏MHC-ICD的结合 到Nef和Clathrin AP1。成功完成这项工作将产生NEF的双功能抑制剂，这 可以用作毒品候选者，以进一步发展为新颖的现实世界抗逆转录病毒，无与伦比 治疗潜力。