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: 10010308
• 负责人: John C. Guatelli
• 金额: $ 20.06万
• 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 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10010308
• 关键词: Acquired Immunodeficiency Syndrome; Adopted; Affinity; Anti-HIV Agents; Anti-Retroviral Agents; Antibodies; 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-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.

项目概要： 目前的抗逆转录病毒药物可以控制艾滋病毒感染，但不能治愈这种疾病。抗艾滋病欠发达地区 药物发现的目的是振兴和利用免疫系统的力量来对抗甚至消除艾滋病毒。 HIV-1 Nef 蛋白调节宿主免疫力，促进疾病进展为艾滋病。两个最重要的 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 相容性复合物（MHC-I）。 Nef 对 CD4 的作用确保了 CD4 的重要组成部分的正确处理 病毒，病毒包膜蛋白。这一行动还有助于病毒隐藏表位并逃避反病毒检测。 体，从而避免由宿主天然介导的抗体依赖性细胞毒性（ADCC） 杀伤细胞用于清除感染。此外，Nef 破坏了另一个重要的免疫识别机制 通过下调 MHC-I。 Nef 的作用是阻断抗原呈递，而抗原呈递是由 MHC-I 介导的 受感染细胞的表面。因此，由于细胞表面缺乏病毒抗原，导致感染 隐藏在免疫监视之下，从而成功地逃避了细胞毒性T细胞的杀伤。鉴于内夫的 由于 Nef 抑制剂具有阻断这些关键免疫机制的能力，因此可以想象，Nef 抑制剂可能会逆转这种机制。 吸吮，这应该能够通过宿主免疫系统检测并可能清除感染。 相信这一假设，我们的目标是通过 Nef 抑制来开发抗逆转录病毒药物，这是我们的长期目标 目标。在这个项目中，我们受到最近结构发现的启发，Nef 使用一个共同的结合位点 执行上述两个函数。这是令人兴奋的，因为我们有可能使用单一药物来 阻断这两种基本功能，可能会恢复巨大的免疫能力来对抗甚至消除艾滋病毒。 我们建议使用并行方法来识别能够与该多功能位点结合的抑制剂 内夫。独特的是，我们以 Nef 可以采用生物活性的方式设计了药物筛选实验。 由于其共同的细胞伙伴，即网格蛋白 AP 复合物的存在，其构象发生了变化。我们的应用程序- Proach 具有创新性，因为我们的设计确保了 Nef 上基材结合口袋的正确形成， 涉及目标多功能站点。我们的具体目标是：1) 识别阻断 CD4 下调的抑制剂 由 Nef 重刑。在这里，我们将使用基于体外荧光偏振的高通量筛选 (HTS) 测定和基于细胞的 CD4 下调测定来寻找阻断 CD4 结合的 Nef 抑制剂 下调； 2) 鉴定可阻断 Nef 下调 MHC-I 的抑制剂。这里我们将使用类似的荧光 基于荧光偏振的 HTS 测定用于筛选可破坏 MHC-ICD 结合的 Nef 抑制剂 Nef 和网格蛋白 AP1。这项工作的成功完成将产生 Nef 双功能抑制剂， 可以作为候选药物进一步开发成具有无与伦比的新型、现实世界抗逆转录病毒药物 治疗潜力。