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.

摘要： 本申请的长期目标是将宿主-病毒相互作用界面表征为新型药物靶标， 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的复制。已经确定，在不影响其酶活性的情况下干扰IN， 抑制HIV-1复制的晚期阶段，例如装配、颗粒产生和/或颗粒形态发生。 这种II类IN突变和IN的变构抑制剂（ALLINI）抑制晚期事件，并且它们通过干扰 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， 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， INI 1/hSNF 5，一种IN结合宿主因子，选择性地掺入HIV-1病毒体。HIV需要INI 1- 1晚事件。INI 1的最小IN结合结构域（INI 1183 -292）（称为S6）的表达破坏了IN/INI 1 在体内相互作用，并有效地抑制HIV-1颗粒的产生。敲除INI 1和INI 1-/-细胞系的使用 抑制HIV-1颗粒产生。INI 1结合缺陷IN突变体导致形态学上的 缺陷颗粒表明靶向IN/INI 1相互作用是抑制HIV-1颗粒的有效策略 生产缺乏INI 1和IN/INI 1复合物的结构已经排除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（Rpt 1）的NMR结构 INI 1的结构域和IN/INI 1相互作用的分子对接有助于克服这一知识差距。 我们发现INI 1的IN结合结构域（Rpt 1）和反式激活反应元件（TAR） HIV-1基因组RNA在结构上相互模仿，这是一个新的发现。Rpt 1和TAR均结合至相同表面 IN C-末端结构域（CTD），并以相同的IC 50值（0.005 µM）竞争与IN结合。 此外，INI 1相互作用缺陷型IN突变体导致颗粒形态发生受损。我们 假设肽模拟物和衍生自Rpt 1小分子具有双重活性并抑制两者 IN/INI 1和IN/TAR相互作用。作为原理的证明，我们已经开发了一种来自于 Rpt 1的界面a-1螺旋，其有效地破坏IN/INI 1和IN/RNA相互作用，抑制颗粒形态发生 和体内HIV-1复制。在这个提议中，我们将IN/INI 1界面描述为一个突出的药物靶点 通过进行：i）遗传分析以了解INI 1对组装/颗粒的影响机制 通过反式互补和“合成拯救”实验生产; ii）开发一类新的 在通过SAR靶向IN/INI 1和IN/RNA相互作用中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INI 1-钉合肽复合物的NMR结构 iii）理解INI 1衍生的钉合肽和小分子 通过筛选病毒逃逸突变体来抑制HIV-1复制和靶向鉴定。这些研究将 确立IN/INI 1作为新药物靶点，并提供新的抑制HIV-1晚期事件的先导化合物。