RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.

RNA模拟引导宿主病毒蛋白的细胞内靶向和病毒RNA-蛋白相互作用以抑制HIV复制。

• 批准号: 10554025
• 负责人: GANJAM V KALPANA
• 金额: $ 61.41万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-06 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10554025
• 关键词: Affect; Anti-HIV Agents; Anti-Retroviral Agents; Antiviral Agents; Binding; Biological Assay; Birds; C-terminal; Cell Line; Cell physiology; Cells; Charge; Collaborations; Complement; Complex; Data; Defect; Development; Docking; Drug Targeting; Drug resistance; Escape Mutant; Event; Genetic; Goals; HIV; HIV-1; HIV-1 integrase; Impairment; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Knowledge; Laboratories; Lead; Libraries; Ligands; Light; Mediating; Molecular; Morphogenesis; Morphology; Mutation; Nature; Nucleic Acids; Peptides; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Positioning Attribute; Production; Protein Engineering; Proteins; RNA; RNA Binding; RNA-Protein Interaction; Reporter; Research; Resistance; Response Elements; Role; SMARCB1 gene; Specificity; Structure; Structure-Activity Relationship; Surface; Testing; Therapeutic; Viral; Viral Proteins; Viral reservoir; Virion; Virus; Virus Latency; Virus Replication; Work; base; design; experimental study; genetic analysis; genomic RNA; improved; in vivo; inhibitor; inorganic phosphate; insight; knock-down; mimicry; mutant; new therapeutic target; novel; particle; peptidomimetics; pharmacophore; pleiotropism; prevent; protein protein interaction; screening; small molecule; stapled peptide; viral RNA; virtual; virus host interaction

Abstract: The long-term goal of this application is to characterize host-virus interaction interface as a novel drug target and to develop inhibitors to disrupt intracellular protein-protein interactions (PPI) between the host and the virus to curb HIV-1 replication. It has been established that perturbing IN without affecting its enzymatic activity can inhibit late stages of HIV-1 replication such as assembly, particle production and/or particle morphogenesis. Such class II IN mutations and allosteric inhibitors of IN (ALLINI), inhibit late events and they do so by perturbing IN/IN multimerization, IN/host factor interaction or IN/RNA interactions. We have observed that such defects in particle morphogenesis can also be observed in IN mutants defective for interaction with a host factor, INI1/hSNF5, an IN-binding host factor, that is selectively incorporated into HIV-1 virions. INI1 is required for HIV- 1 late events. Expression of a minimal-IN-binding domain of INI1 (INI1183-292) termed S6, disrupts IN/INI1 interaction in vivo and potently inhibits HIV-1 particle production. Knocking down INI1 and use of INI1-/- cell lines inhibit HIV-1 particle production. INI1-binding defective IN mutants lead to the production of morphologically defective particles indicating that targeting IN/INI1 interaction is an effective strategy to inhibit HIV-1 particle production. Lack of structure of INI1 and IN/INI1 complex have precluded our ability to develop inhibitors to target this interaction. New research from our laboratory including the NMR structure of the IN-binding Repeat 1 (Rpt1) domain of INI1, and molecular docking of IN/INI1 interaction have helped overcome this knowledge gap. We found that IN-binding domain of INI1, termed Rpt1, and Trans Activating Response element (TAR) of HIV-1 genomic RNA structurally mimic each other, a novel finding. Both Rpt1 and TAR bind to same surface of IN C-terminal domain (CTD) and compete for binding to IN with an identical IC50 value (0.005 µM). Furthermore, INI1-interaction-defective mutants of IN cause impairment of particle morphogenesis. We hypothesize that peptidomimetics and small molecules derived from Rpt1 have dual activity and inhibit both IN/INI1 and IN/TAR interactions. As a proof of principle, we have developed a stapled peptide derived from interface a-1 helix of Rpt1, that potently disrupts IN/INI1 and IN/RNA interactions, inhibits particle morphogenesis and in vivo HIV-1 replication. In this proposal, we will characterize IN/INI1 interface as an outstanding drug target by carrying out: i) Genetic analysis to understand the mechanism of INI1 influence on assembly/particle production via trans-complementation and “synthethic rescue” experiments; ii) Development of a novel class of stapled peptides and small molecules with dual activity in targeting IN/INI1 and IN/RNA interactions by SAR and virtual-ligand screening; and determine the NMR structure of INI1-stapled peptide complexes with IN-C-terminal domain; and iii) Understanding the mechanism by which INI1-derived stapled peptides and small molecules inhibit HIV-1 replication and target identification by screening for viral escape mutants. These studies will establish IN/INI1 as a novel drug target and provide new lead compounds to inhibit HIV-1 late events.

抽象的： 该应用的长期目标是将宿主-病毒相互作用界面表征为新的药物靶点并 开发抑制剂来破坏宿主和病毒之间的细胞内蛋白质-蛋白质相互作用（PPI） 抑制 HIV-1 复制。已经证实，在不影响 IN 酶活性的情况下扰动 IN 可以 抑制 HIV-1 复制的后期阶段，例如组装、颗粒产生和/或颗粒形态发生。 此类 II 类 IN 突变和 IN 变构抑制剂 (ALLINI) 可抑制晚期事件，并且它们通过扰动来抑制晚期事件 IN/IN 多聚化、IN/宿主因子相互作用或 IN/RNA 相互作用。我们观察到，此类缺陷 在与宿主因子相互作用有缺陷的 IN 突变体中也可以观察到颗粒形态发生， INI1/hSNF5 是一种 IN 结合宿主因子，选择性掺入 HIV-1 病毒颗粒中。 HIV 需要 INI1- 1 迟到的事件。 INI1 (INI1183-292) 的最小 IN 结合域（称为 S6）的表达会破坏 IN/INI1 体内相互作用并有效抑制 HIV-1 颗粒的产生。敲低 INI1 并使用 INI1-/- 细胞系 抑制 HIV-1 颗粒的产生。 INI1 结合缺陷 IN 突变体导致形态学上的产生 有缺陷的颗粒表明靶向 IN/INI1 相互作用是抑制 HIV-1 颗粒的有效策略 生产。 INI1 和 IN/INI1 复合物结构的缺乏阻碍了我们开发靶向抑制剂的能力 这种互动。我们实验室的新研究，包括 IN 结合重复序列 1 (Rpt1) 的 NMR 结构 INI1 的结构域以及 IN/INI1 相互作用的分子对接有助于克服这一知识差距。 我们发现 INI1 的 IN 结合域（称为 Rpt1）和反式激活响应元件（TAR） HIV-1 基因组 RNA 的结构相互模仿，这是一项新发现。 Rpt1 和 TAR 都结合到同一表面 IN C 端结构域 (CTD) 并以相同的 IC50 值 (0.005 µM) 竞争与 IN 的结合。 此外，INI1 相互作用缺陷突变体会导致颗粒形态发生受损。我们 假设肽模拟物和源自 Rpt1 的小分子具有双重活性并抑制两者 IN/INI1 和 IN/TAR 交互。作为原理证明，我们开发了一种来自以下物质的钉合肽： Rpt1 的界面 a-1 螺旋，有效破坏 IN/INI1 和 IN/RNA 相互作用，抑制颗粒形态发生 和体内 HIV-1 复制。在本提案中，我们将把 IN/INI1 接口描述为一个杰出的药物靶点 通过执行： i) 遗传分析来了解 INI1 对组装/粒子的影响机制 通过反式互补和“合成救援”实验进行生产； ii) 开发一类新的 通过 SAR 和靶向 IN/INI1 和 IN/RNA 相互作用具有双重活性的钉合肽和小分子 虚拟配体筛选；并确定具有 IN-C-末端的 INI1-stapled 肽复合物的 NMR 结构 领域; iii) 了解 INI1 衍生的钉合肽和小分子的机制 通过筛选病毒逃逸突变体来抑制 HIV-1 复制和靶标识别。这些研究将 建立 IN/INI1 作为新的药物靶点，并提供新的先导化合物来抑制 HIV-1 晚期事件。