Structure-based design of stapled peptides to target Gag-Pol and INI1 interaction to block assembly

基于结构的钉合肽设计，以靶向 Gag-Pol 和 INI1 相互作用来阻止组装

• 批准号: 10302316
• 负责人: GANJAM V KALPANA
• 金额: $ 21.13万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-13 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302316
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Amino Acids; Binding; Biochemical; Biological Assay; Biological Products; Birds; Blood; C-terminal; Cell Line; Cells; Charge; Collaborations; Development; Docking; Electron Microscopy; Electrostatics; Event; Fluorescence Resonance Energy Transfer; Goals; HIV; HIV-1; HIV-1 integrase; Hydrocarbons; Impairment; In Vitro; Infection; Integrase; Integrase Inhibitors; Integration Host Factors; Knowledge; Laboratories; Lead; Modeling; Molecular; Molecular Mimicry; Morphogenesis; Morphology; Mutation; Nature; New York; Nucleic Acids; Penetration; Peptides; Peripheral Blood Mononuclear Cell; Positioning Attribute; Production; Property; Proteins; Proteolysis; Quality Control; RNA; Reaction; Reagent; Reporter; Reporting; Resistance; Response Elements; Role; SMARCB1 gene; Series; Structure; Surface; Testing; Therapeutic; Toxic effect; Triazoles; Viral; Viral Proteins; Virus; Virus Replication; alpha helix; base; cycloaddition; design; genomic RNA; in vivo; inhibitor; inorganic phosphate; insight; knock-down; mimicry; mutant; novel; novel strategies; novel therapeutics; pandemic disease; particle; peptidomimetics; protein protein interaction; response; stapled peptide; success; therapeutic target

Abstract: This application is in response to RFA-AI-19-072, “Novel Therapeutics Directed to Intracellular HIV Targets”. The long term goal of this application is to develop stapled peptide inhibitors to disrupt intracellular protein-protein interactions (PPI) between the host and the virus to curb HIV-1 replication. PPI surfaces are hard to disrupt because of their large and flat surface of interactions. However, recent success in the development of larger biologics such as hydrocarbon stapled peptides allows targeting of PPIs. The stapled peptides are a helices from binding interfaces of PPI that are locked into their bioactive forms. Our goal is to intracellularly disrupt HIV-1 integrase (IN) interaction with the host factor INI1/hSNF5 using stapled peptides, to inhibit HIV-1 assembly, particle production and/or particle morphogenesis. 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. Several 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. INI1/hSNF5 is the first IN-binding host factor to be identified. We have extensively studied its role in HIV-1 replication and found that it is required for HIV-1 late events. We found that 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 also inhibit HIV-1 particle production. Interestingly, IN mutants that are defective for binding to INI1 lead to the production of morphologically defective particles. These studies together indicate that targeting IN/INI1 interaction is an effective strategy to inhibit HIV-1 particle production. However, lack of structure of INI1 and IN/INI1 interactions have precluded our ability to develop inhibitors to target this interaction. Recent developments in our laboratory in solving the NMR structure of the IN-binding Repeat 1 (Rpt1) domain of INI1, and molecular docking studies of IN/INI1 interaction have helped to overcome this knowledge gap. These structural studies have been validated by mutational, biochemical and virological studies that establish the significance of IN/INI1 interactions. During our structural studies we made an unprecedented novel discovery that INI1 Rpt1 and Trans Activating Response element (TAR) of HIV-1 genomic RNA structurally mimic each other. Nucleic acid mimicry by proteins exists in nature, but mimicry of Rpt1 to TAR is novel and has not been reported earlier. We found that both Rpt1 and TAR bind to same surface of IN C-terminal domain (CTD) and compete with each other for binding to IN with identical IC50 value of 0.005 µM. Furthermore, INI1-interaction-defective mutants of IN resulted in impairment of particle morphogenesis, indicating that these mutants do not bind to RNA in vivo. The knowledge about structural mimicry between Rpt1 and TAR have provided novel strategies to target these interactions. Based on the fact that Rpt1 domain disrupts both IN/INI1 and IN/TAR interactions, we hypothesize that peptidomimetics derived from Rpt1 have dual activity and inhibit both IN/INI1 and IN/TAR interactions. Thus, designing inhibitors using IN/INI1 interaction have the benefit of “killing two birds in one stone”. This proposal is in collaboration with a medicinal chemist Dr. Asim Debnath (New York Blood Center). In Aim I we will design INI1-based antiviral peptides with enhanced α-helicity, cell-penetrating properties, and resistance against proteolysis through peptide stapling. In aim II we will test the effect of Stapled peptides on IN-INI1, IN-RNA interactions and on HIV-1 replication: These studies are likely to yield novel stapled dual-active peptides that target intracellular IN/INI1 and/or IN/RNA interactions to inhibit HIV-1 late events.

摘要： 本申请是对RFA-AI-19-072“针对细胞内HIV的新型治疗药物”的回应 目标”。本申请的长期目标是开发钉合肽抑制剂，以破坏细胞内的细胞毒性。 蛋白质-蛋白质相互作用（PPI）之间的主机和病毒，以遏制HIV-1复制。PPI表面坚硬 因为它们大而平的相互作用表面。然而，最近在开发 更大的生物制剂如烃钉合肽允许PPI的靶向。钉合肽是一种 来自PPI结合界面的螺旋被锁定成其生物活性形式。我们的目标是在细胞内 使用钉合肽破坏HIV-1整合酶（IN）与宿主因子INI 1/hSNF 5的相互作用，以抑制HIV-1 组装、颗粒产生和/或颗粒形态发生。 已经确定，干扰IN而不影响其酶活性可以抑制晚期的 HIV-1复制，如组装、颗粒产生和/或颗粒形态发生。几个II类IN IN的突变和变构抑制剂（ALLINI）抑制晚期事件，并且它们通过干扰IN/IN来实现 多聚化、IN/宿主因子相互作用或IN/RNA相互作用。INI 1/hSNF 5是第一个IN结合宿主因子 待鉴定我们广泛研究了它在HIV-1复制中的作用，发现它是HIV-1所必需的， 后期事件。我们发现INI 1的最小IN结合结构域（INI 1183 -292）的表达，称为S6， IN/INI 1相互作用，并有效抑制HIV-1颗粒的产生。敲除INI 1和使用INI 1-/- 细胞系也抑制HIV-1颗粒的产生。有趣的是，与INI 1结合有缺陷的IN突变体导致 涉及形态缺陷颗粒的生产。这些研究共同表明，靶向IN/INI 1 相互作用是抑制HIV-1颗粒产生有效策略。然而，由于缺乏国家信息网的结构， IN/INI 1相互作用已经排除了我们开发针对这种相互作用的抑制剂的能力。最近 我们实验室在解决INI 1的IN结合重复1（Rpt 1）结构域的NMR结构方面的进展， IN/INI 1相互作用的分子对接研究有助于克服这一知识差距。这些 结构研究已经通过突变、生物化学和病毒学研究得到验证， IN/INI 1相互作用的重要性。 在我们的结构研究中，我们发现了一个前所未有的新发现，INI 1 Rpt 1和Trans HIV-1基因组RNA的激活反应元件（TAR）在结构上相互模仿。核酸模拟 天然存在的蛋白质，但Rpt 1对TAR的模拟是新的，以前没有报道。我们发现 Rpt 1和TAR均与IN C-末端结构域（CTD）相同表面结合， 与IN结合，IC 50值相同，为0.005 µM。此外，INI 1相互作用缺陷的IN突变体导致 在颗粒形态发生的损害，表明这些突变体不结合RNA在体内。知识 关于Rpt 1和TAR之间的结构模拟的研究提供了针对这些相互作用的新策略。 基于Rpt 1结构域破坏IN/INI 1和IN/TAR相互作用的事实，我们假设， 衍生自Rpt 1的肽模拟物具有双重活性并抑制IN/INI 1和IN/TAR相互作用。因此，在本发明中， 利用IN/INI 1相互作用设计抑制剂具有“一石二鸟”的好处。这项建议是 与药物化学家Asim Debnath博士（纽约血液中心）合作。在Aim I中，我们将设计 基于INI 1的抗病毒肽具有增强的α-螺旋性、细胞穿透性和抗 通过肽钉合进行蛋白水解。在目的II中，我们将测试钉合肽对IN-INI 1、IN-RNA 相互作用和HIV-1复制：这些研究可能产生新的钉合双活性肽， 靶向细胞内IN/INI 1和/或IN/RNA相互作用以抑制HIV-1晚期事件。