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

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无法清除 HIV-1 潜伏病毒库，这凸显了迫切需要 新的治疗策略。 HIV-1 Nef 辅助因子是药物开发的一个有吸引力的目标，因为 其在 HIV-1 生命周期和免疫系统逃逸中的关键作用。我们小组发现了新奇的小 直接与 Nef 结合并阻断其许多功能的分子，包括增强病毒感染性和 在供体 PBMC 中复制。重要的是，我们的 Nef 抑制剂可以挽救细胞表面 MHC-I 的潜在表达。 感染的、患者来源的 CD4+ T 细胞，能够被自体 CTL 识别和杀死。因此，内夫 抑制剂代表了一种创新的抗逆转录病毒治疗方法，可能提供根除病毒的途径 病毒储存库。我们最有前途的一类抑制剂（羟基吡唑）与其 Nef 蛋白靶点紧密结合 在体外，对基于细胞的系统中的多种 Nef 功能具有活性，且无细胞毒性。实验 这里提出的将利用这些化合物作为化学探针，为 Nef 功能提供新的线索，同时 通过以下具体目标阐明其作用机制： 目标 1. 绘制羟基的结合位点图 吡唑 Nef 抑制剂的 X 射线晶体学分析。初步和已发表的数据强烈表明羟基 吡唑 Nef 抑制剂会破坏多种 Nef 功能，可能会扰乱功能性 Nef 效应器的结构 复合物。单独使用 Nef 以及与宿主细胞效应蛋白复合的抑制剂的 X 射线晶体学 将用于测试这个想法并确定抑制剂结合位点。目标 2. 鉴定必需的 Nef 残基 通过体外选择产生抑制剂作用。使用基于 PCR 的饱和诱变，我们替换了所有 HIV-1 中 Nef 核心区的密码子与 64 个核苷酸三联体中的每一个。 CD4 T 细胞将 在存在或不存在 Nef 抑制剂的情况下感染 Nef 突变病毒“文库”以及病毒上清液 通过深度测序进行分析，以确定通过抑制剂治疗富集的突变。这种方法有潜力 除了直接参与配体的残基之外，还可以识别以变构方式影响抑制剂作用的 Nef 区域 绑定。目标 3. 探索 Nef 抑制剂抑制 HIV-1 感染性的机制。羟基吡唑 Nef 抑制剂降低 TZM-bl 报告细胞中 HIV-1 感染性的程度与 Nef 缺失病毒相同。这 Aim 将探索 Nef 抑制剂是否可以恢复病毒粒子对两种宿主 SERINC 蛋白和 Ezrin 的掺入 与 Nef 相关的细胞限制因子。我们还将研究 Nef 抑制剂对 HIV-1 蛋白质整体组成的影响 通过全病毒蛋白质组学，它有可能识别独特整合的宿主细胞因子（或 排除）通过 Nef 抑制。目标 4. 研究 Nef 抑制剂对 MHC-I 下调的作用机制。 该目的将探讨 Nef 抑制剂对 Nef 与 MHC-I 细胞复合物晶体结构的影响。 质尾和 AP-1 µ1 亚基，相互作用对于免疫逃逸至关重要。 Nef 相互作用的抑制剂作用 还将利用双分子荧光互补 (BiFC) 在细胞中探索 MHC-I 和 AP-1 的相互作用。 这些研究将阐明 Nef 抑制剂恢复 CTL 对 HIV 感染反应的机制。