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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结: