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.

抽象的： 该应用是响应RFA-AI-19-072，“针对细胞内HIV的新型治疗学 目标”。该应用的长期目标是开发滞留的肽抑制剂以破坏细胞内 宿主与病毒之间的蛋白质蛋白相互作用（PPI），以遏制HIV-1复制。 PPI表面很难 由于相互作用的较大而平坦的表面而破坏。但是，最近在发展方面取得了成功 较大的生物制剂，例如碳氢化合物阶段的肽允许靶向PPI。上演的肽是 PPI结合界面的螺旋螺旋锁定在其生物活性形式中。我们的目标是细胞内 使用Stagned肽破坏与宿主因子INI1/HSNF5相互作用的HIV-1积分酶（IN）相互作用，以抑制HIV-1 组装，颗粒产生和/或粒子形态发生。 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，例如组装，颗粒产生和/或粒子形态发生。几个II类 IN（Allini）的突变和变构抑制剂，抑制后期事件，它们通过扰动/in in in/in 多层化，/宿主因子相互作用或/RNA相互作用。 INI1/HSNF5是第一个内在宿主因子 被识别。我们已经广泛研究了其在HIV-1复制中的作用，发现HIV-1是必需的 晚事。我们发现INI1（INI1183-292）的最小结合域的表达称为S6 在体内/INI1相互作用中，可能会抑制HIV-1颗粒的产生。击倒INI1并使用INI1 - / - 细胞系还抑制HIV-1颗粒的产生。有趣的是，在与INI1铅结合有缺陷的突变体中 生产形态上有缺陷的颗粒。这些研究一起表明针对/INI1的靶向 相互作用是抑制HIV-1颗粒产生的有效策略。但是，缺乏INI1和 在/INI1相互作用中，我们排除了我们开发抑制剂靶向这种相互作用的能力。最近的 我们实验室的发展解决了INI1的内在重复1（RPT1）域的NMR结构， IN/INI1相互作用的分子对接研究有助于克服这一知识差距。这些 结构研究已通过突变，生化和病毒学研究来验证 IN/INI1相互作用的意义。 在我们的结构研究中，我们做出了一个空前的新发现，即Ini1 RPT1和Trans 激活HIV-1基因组RNA的反应元件（tar）在结构上模仿。核酸模仿 自然界中存在蛋白质，但是RPT1对TAR的模仿是新颖的，尚未较早报道。我们发现 RPT1和TAR都与C末端结构域（CTD）中的同一表面结合，并相互竞争 与IC50值相同的0.005 µm结合。此外，Ini1相互作用缺陷突变体导致 在损伤的颗粒形态发生时，表明这些突变体在体内不与RNA结合。知识 关于RPT1和TAR之间的结构模仿，已经提供了针对这些相互作用的新型策略。 基于RPT1域在/INI1和/TAR相互作用中都破坏了这一事实，我们假设 源自RPT1的肽仪具有双重活性，并且在/INI1和/tar相互作用中都抑制。那， 使用/INI1相互作用的设计抑制剂具有“用一块石头杀死两只鸟”的好处。该提议是 与医学化学家Asim Debnath（纽约血液中心）合作。在目的中，我将设计 基于INI1的抗病毒肽具有增强的α-螺旋，细胞渗透特性和抵抗力 蛋白水解通过肽钉钉。在AIM II中，我们将测试钉肽对INI1，RNA的影响 相互作用和HIV-1复制：这些研究可能会产生新的分阶性双重活动宠物 靶向细胞内/INI1和/或/或/或/或RNA相互作用，以抑制HIV-1后期事件。