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控制艾滋病毒感染，但不能治愈这种疾病。一个抗艾滋病不发达的地区 药物发现的目的是恢复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以促进疾病进展为AIDS。两个最重要的- Nef的重要功能是表面下调CD 4和表面下调I类主要组织蛋白， 相容性复合物（MHC-I）。Nef对CD 4的作用确保了对CD 4的一个重要组成部分的正确处理。 病毒的包膜蛋白这种作用还有助于病毒隐藏表位并逃避抗- 抗体依赖性细胞毒性（ADCC），这是由宿主的天然抗体介导的。 清除感染的杀伤细胞此外，Nef还破坏了另一种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，抗原呈递是由MHC-I介导的。 感染细胞的表面。因此，由于细胞表面缺乏病毒抗原， 隐藏在免疫监视之外，从而成功逃避细胞毒性T细胞的杀伤。关于Nef 阻止这些关键免疫机制的能力，可以想象Nef的抑制剂可以逆转这种疾病。 nipulation，这应该能够检测和可能清除感染的宿主免疫系统。 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，我们的灵感来自于我们最近的结构发现，Nef使用一个共同的结合位点， 执行上述两个功能。这是令人兴奋的，因为我们可能使用一种药物， 阻断这两种基本功能，这可能会恢复巨大的免疫力，以对抗甚至消除艾滋病毒。 我们建议使用一种新的方法来识别能够结合到这个多功能位点的抑制剂， 内夫独特的是，我们设计了我们的药物筛选实验的方式，Nef可以采用生物活性， 活性构象，由于其存在的增选细胞的合作伙伴，即网格蛋白AP复合物。我们的ap- 该方法是创新的，因为我们的设计确保了Nef上的基底结合口袋的正确形成， 涉及目标多功能网站。我们的具体目标是：1）确定阻断CD 4下调的抑制剂- 的Nef。在这里，我们将使用体外荧光偏振为基础的，高通量筛选（HTS） 测定和基于细胞的CD 4下调测定以寻找阻断CD 4结合的Nef抑制剂， 下调; 2）鉴定阻断由Nef引起的MHC-I下调的抑制剂。在这里，我们将使用类似的透视- 用于筛选可破坏MHC-ICD结合的Nef抑制剂的基于光偏振的HTS测定 Nef和网格蛋白AP 1。这项工作的成功完成将产生双功能Nef抑制剂， 可以作为候选药物进一步开发成新的、真实的世界抗逆转录病毒药物， 治疗潜力