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一种新型药物靶标，并且 开发抑制剂以破坏宿主和病毒之间的细胞内蛋白质蛋白质相互作用（PPI） 遏制HIV-1复制。已经确定，在不影响其酶活性的情况下扰动可以 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类在突变和in（allini）的变构抑制剂中，抑制后期事件，它们通过扰动来做到这一点 在/宿主因子相互作用或/rNA相互作用中/在多层化中。我们观察到此类缺陷在 在突变体中也可以观察到粒子形态发生，以与宿主因子相互作用， INI1/HSNF5是一种结合宿主因子，被选择地纳入HIV-1病毒。 Ini1是艾滋病毒所必需的 1个晚事。 INI1（INI1183-292）的最小结合域的表达称为S6，在/INI1中破坏 体内相互作用并可能抑制HIV-1颗粒的产生。拆卸INI1并使用INI1 - / - 细胞系 抑制HIV-1颗粒产生。突变体中Ini1结合缺陷导致形态学产生 有缺陷的颗粒表明靶向/INI1相互作用是抑制HIV-1粒子的有效策略 生产。缺乏INI1和INI1复合物的结构，已经阻止了我们开发抑制剂靶向的能力 这种相互作用。我们实验室的新研究，包括内在重复1（RPT1）的NMR结构 INI1的域和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的内在结构域称为RPT1，而反式激活响应元件（TAR） HIV-1基因组RNA在结构上彼此模仿，这是一种新颖的发现。 RPT1和焦油都结合到同一表面 在C末端结构域（CTD）中，与IC50值相同（0.005 µM）竞争与IN结合。 此外，INI1相互作用缺陷突变体导致颗粒形态发生损害。我们 假设从RPT1得出的辣妹和小分子具有双重活性，并且抑制 在/ini1和/tar相互作用。作为原则的证明，我们已经开发了一种衍生出的分阶段肽 RPT1的接口A-1螺旋螺旋可能会破坏/INI1和/RNA相互作用，抑制粒子形态发生 和体内HIV-1复制。在此提案中，我们将在/INI1接口中将其描述为未出色的药物目标 通过进行：i）遗传分析以了解INI1对组装/粒子的影响的机制 通过反式融合和“合成救援”实验生产； ii）开发新颖的一类 固定肽和小分子在靶向/INI1和/RNA相互作用中具有双重活性的小分子和SAR和RNA相互作用 虚拟配体筛选；并确定INI1 stapled肽复合物与C末端的NMR结构 领域; iii）了解INI1衍生的毒性肽和小分子的机制 通过筛选病毒逃生突变体来抑制HIV-1复制和靶标识别。这些研究会 在/INI1中建立作为一种新型药物靶标，并提供新的铅化合物来抑制HIV-1后期事件。