Chemical Biology of HIV-1 Nef

HIV-1 Nef 的化学生物学

• 批准号: 10251040
• 负责人: Thomas E. Smithgall
• 金额: $ 61.93万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10251040
• 关键词: 3-Dimensional; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Affect; Anti-Retroviral Agents; Autologous; Binding; Binding Sites; Biological Assay; Biology; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Surface Receptors; Cell surface; Cells; Chemicals; Codon Nucleotides; Complement; Complex; Crystallization; Crystallography; Cytotoxic T-Lymphocytes; Data; Development; Down-Regulation; Drug Targeting; Effector Cell; Fluorescence; HIV; HIV Infections; HIV-1; Immune; Immune system; In Vitro; Infection; Laboratories; Libraries; Life; Life Cycle Stages; Ligand Binding; Light; Link; Maps; Mediating; Methods; Molecular Conformation; Mutagenesis; Mutation; Nucleotides; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Protein Tyrosine Kinase; Proteins; Proteomics; Publishing; Pyrazoles; Relapse; Reporter; Reporting; Roentgen Rays; Role; SH3 Domains; Structure; Surface; System; Tail; Testing; Therapeutic; Transcription Factor AP-1; Triplet Multiple Birth; Viral; Viral reservoir; Virion; Virus; Virus Replication; Work; X-Ray Crystallography; antiretroviral therapy; base; cell killing; clinical translation; cytotoxicity; deep sequencing; drug candidate; drug development; experimental study; ezrin; in vivo; inhibitor/antagonist; innovation; insight; latent HIV reservoir; mutant; nef Protein; novel; novel strategies; novel therapeutic intervention; prevent; protein complex; receptor downregulation; recruit; response; small molecule; src Homology Region 2 Domain; src-Family Kinases; therapeutic target; virtual; 3-Dimensional; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Affect; Anti-Retroviral Agents; Autologous; Binding; Binding Sites; Biological Assay; Biology; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Surface Receptors; Cell surface; Cells; Chemicals; Codon Nucleotides; Complement; Complex; Crystallization; Crystallography; Cytotoxic T-Lymphocytes; Data; Development; Down-Regulation; Drug Targeting; Effector Cell; Fluorescence; HIV; HIV Infections; HIV-1; Immune; Immune system; In Vitro; Infection; Laboratories; Libraries; Life; Life Cycle Stages; Ligand Binding; Light; Link; Maps; Mediating; Methods; Molecular Conformation; Mutagenesis; Mutation; Nucleotides; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Protein Tyrosine Kinase; Proteins; Proteomics; Publishing; Pyrazoles; Relapse; Reporter; Reporting; Roentgen Rays; Role; SH3 Domains; Structure; Surface; System; Tail; Testing; Therapeutic; Transcription Factor AP-1; Triplet Multiple Birth; Viral; Viral reservoir; Virion; Virus; Virus Replication; Work; X-Ray Crystallography; antiretroviral therapy; base; cell killing; clinical translation; cytotoxicity; deep sequencing; drug candidate; drug development; experimental study; ezrin; in vivo; inhibitor/antagonist; innovation; insight; latent HIV reservoir; mutant; nef Protein; novel; novel strategies; novel therapeutic intervention; prevent; protein complex; receptor downregulation; recruit; response; small molecule; src Homology Region 2 Domain; src-Family Kinases; therapeutic target; virtual

Summary. Existing antiretroviral drugs do not clear HIV-1 latent reservoirs, underscoring the urgent need for new therapeutic strategies. The HIV-1 Nef accessory factor is an attractive target for drug development because of its critical roles in the HIV-1 life cycle and immune system escape. Our group has discovered novel small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in donor PBMCs. Importantly, our Nef inhibitors rescue cell-surface MHC-I expression in latently infected, patient-derived CD4+ T-cells, enabling recognition and killing by autologous CTLs. Thus, Nef inhibitors represent an innovative approach to antiretroviral therapy that may provide a path to eradication of viral reservoirs. Our most promising class of inhibitors (hydroxypyrazoles) bind tightly to their Nef protein target in vitro and are active against multiple Nef functions in cell-based systems without cytotoxicity. Experiments proposed here will leverage these compounds as chemical probes to shed new light on Nef functions while unraveling their mechanism of action with the following Specific Aims: Aim 1. Map the binding site for hydroxy- pyrazole Nef inhibitors by X-ray crystallography. Preliminary and published data strongly suggest that hydroxy- pyrazole Nef inhibitors, which disrupt multiple Nef functions, may perturb the structure of functional Nef-effector complexes. X-ray crystallography of inhibitors with Nef alone and in complexes with host cell effector proteins will be used to test this idea and identify inhibitor binding sites. Aim 2. Identification of Nef residues essential for inhibitor action through in vitro selection. Using PCR-based saturation mutagenesis, we have replaced every codon in the Nef core region with each of the 64 nucleotide triplets in the context of HIV-1. CD4 T cells will be infected with the Nef mutant viral ‘library’ in the presence or absence of Nef inhibitors, and viral supernatants analyzed by deep sequencing to identify mutations enriched by inhibitor treatment. This method has the potential to identify Nef regions that allosterically influence inhibitor action in addition to residues directly involved in ligand binding. Aim 3. Explore the mechanisms by which Nef inhibitors suppress HIV-1 infectivity. Hydroxypyrazole Nef inhibitors reduce HIV-1 infectivity in TZM-bl reporter cells to the same extent as Nef-deleted viruses. This Aim will explore the whether Nef inhibitors restore virion incorporation of SERINC proteins and Ezrin, two host cell restriction factors linked to Nef. We will also pursue Nef inhibitor effects on overall HIV-1 protein composition by whole-virus proteomics, which has the potential to identify host cell factors that are uniquely incorporated (or excluded) by Nef inhibition. Aim 4. Investigate the mechanism of Nef inhibitor action on MHC-I downregulation. This Aim will explore the effect of Nef inhibitors on crystal structures of Nef in complexes with the MHC-I cyto- plasmic tail and the AP-1 µ1 subunit, interactions essential for immune escape. Inhibitor effects on Nef interac- tions with MHC-I and AP-1 will also be explored in cells using bimolecular fluorescence complementation (BiFC). These studies will clarify the mechanisms by which Nef inhibitors restore CTL responses to HIV infection.

概括。现有的抗逆转录病毒药物尚未清除HIV-1潜在储层，强调了迫切需要 新理论策略。 HIV-1 NEF附件因子是药物开发的有吸引力的目标，因为 它在HIV-1生命周期和免疫系统中逃脱的关键作用。我们的小组发现了小说 直接与NEF结合并阻止其许多功能的分子，包括增强病毒感染和 供体PBMC中的复制。重要的是，我们的NEF抑制剂挽救细胞表面MHC-I在潜在的 被感染的，患者来源的CD4+ T细胞，可自体CTL识别并杀死。那，nef 抑制剂代表一种抗逆转录病毒疗法的创新方法，该方法可能提供消除的途径 病毒水库。我们最有前途的抑制剂（羟基吡唑）与NEF蛋白靶标紧密结合 体外，对基于细胞的系统的多个NEF功能具有活性，而没有细胞毒性。实验 这里提出的将利用这些化合物作为化学问题，从而为NEF功能提供新的启示 用以下特定目的阐明其作用机理：目标1。绘制羟基的结合位点 吡喃唑NEF抑制剂通过X射线晶体学。初步和发布的数据强烈表明羟基 - 破坏多个NEF功能的吡唑NEF抑制剂可能会扰动功能NEF-effter的结构 复合物。单独使用NEF和具有宿主细胞效应蛋白的复合物的抑制剂的X射线晶体学 将用于测试这一想法并识别抑制剂结合位点。目标2。确定NEF残基必不可少的 通过体外选择的抑制剂作用。使用基于PCR的满意诱变，我们替换了每个 在HIV-1的背景下，在NEF核心区域中的密码子与64个核苷酸三联体中的每个密码子。 CD4 T细胞将是 在存在或不存在NEF抑制剂和病毒上清液的情况下，被NEF突变病毒“文库”感染 通过深度测序分析以鉴定通过抑制剂治疗富集的突变。该方法具有潜力 确定除了直接参与配体的抑制剂作用会影响抑制剂作用的NEF区域 结合。目标3。探索NEF抑制剂抑制HIV-1感染的机制。羟型吡唑 NEF抑制剂降低了TZM-BL报告细胞中HIV-1感染的程度与NEF删除病毒相同。这 AIM将探索NEF抑制剂是否恢复两个宿主的序列蛋白和Ezrin的病毒体保险 与NEF相关的细胞限制因子。我们还将追求NEF抑制剂对总体HIV-1蛋白组成的影响 通过全病毒蛋白质组学，它有可能识别唯一纳入的宿主细胞因子（或 不包括NEF抑制。目标4。研究NEF抑制剂作用对MHC-I下调的机制。 该目标将探索NEF抑制剂对MHC-I Cyto-复合物中NEF晶体结构的影响 等离子尾和AP-1 µ1亚基，对免疫逃生必不可少的相互作用。抑制剂对NEF间间的影响 使用双分子荧光完成（BIFC）在细胞中也将探索使用MHC-I和AP-1的TION。 这些研究将阐明NEF抑制剂恢复CTL对HIV感染的反应的机制。