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.

摘要现有的抗逆转录病毒药物不能清除HIV-1潜伏宿主，这突出表明迫切需要 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靶点， 它在HIV-1生命周期和免疫系统逃逸中的关键作用。我们小组发现了一种新的小型 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， 在供体PBMC中复制。重要的是，我们的Nef抑制剂在潜伏期内拯救细胞表面MHC-I表达。 感染的患者来源的CD 4 + T细胞，能够被自体CTL识别和杀伤。因此，Nef 抑制剂代表了抗逆转录病毒疗法的一种创新方法， 病毒储存库。我们最有前途的一类抑制剂（羟基吡唑类）与其Nef蛋白靶点紧密结合 并且在基于细胞的系统中对多种Nef功能有活性而无细胞毒性。实验 本文提出的方法将利用这些化合物作为化学探针，对Nef功能进行新的研究， 阐明其作用机制，具体目标如下：目标1。绘制羟基- 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- 吡唑类Nef抑制剂可破坏多种Nef功能， 配合物Nef单独和与宿主细胞效应蛋白复合的抑制剂的X射线晶体学 将被用来测试这个想法，并确定抑制剂结合位点。目标2. Nef残基的鉴定 通过体外选择抑制剂作用。使用基于PCR的饱和诱变，我们已经取代了每一个 在HIV-1的情况下，Nef核心区的密码子与64个核苷酸三联体中的每一个。CD 4 T细胞是 在存在或不存在Nef抑制剂的情况下用Nef突变体病毒“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。这种方法有潜力 为了鉴定除了直接参与配体的残基之外，变构影响抑制剂作用的Nef区域， 约束力目标3.探索Nef抑制剂抑制HIV-1感染的机制。羟基吡唑 Nef抑制剂降低TZM-bl报告细胞中的HIV-1感染性的程度与Nef缺失病毒相同。这 目的将探索Nef抑制剂是否恢复病毒粒子掺入SERINC蛋白和Ezrin，两个宿主 与Nef相关的细胞限制因子。我们还将研究Nef抑制剂对HIV-1蛋白组成的影响 通过全病毒蛋白质组学，它有可能识别独特掺入的宿主细胞因子（或 排除）通过Nef抑制。目标4。探讨Nef抑制剂对MHC-I下调的作用机制。 本研究旨在探索Nef抑制剂对Nef与MHC-I细胞因子复合物中Nef晶体结构的影响。 plasma tail和AP-1 µ1亚基，免疫逃逸所必需的相互作用。抑制剂对Nef相互作用 还将使用双分子荧光互补（BiFC）在细胞中探索MHC-I和AP-1的作用。 这些研究将阐明Nef抑制剂恢复HIV感染的CTL应答的机制。