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

摘要： 该应用是对RFA-AI-19-072的响应，该RFA-AI-19-072是针对细胞内HIV的新型治疗药物 目标“。这一应用的长期目标是开发能干扰细胞内的多肽抑制物 宿主和病毒之间的蛋白质-蛋白质相互作用(PPI)，以抑制HIV-1复制。PPI表面很硬 因为它们相互作用的表面很大很平坦，所以会受到干扰。然而，最近在开发方面的成功 更大的生物制剂，如碳氢化合物装订的多肽，可以靶向PPI。装订的多肽是一种 来自PPI结合界面的螺旋被锁定为其生物活性形式。我们的目标是在细胞内 用装订的多肽阻断HIV-1整合酶(IN)与宿主因子INI1/hSNF5的相互作用，抑制HIV-1 组装、颗粒生产和/或颗粒形态发生。 已经证实，在不影响其酶活性的情况下干扰IN可以抑制晚期的 HIV-1复制，如组装、颗粒生产和/或颗粒形态形成。几个II类IN IN(Allini)的突变和变构抑制剂抑制晚期事件，它们通过干扰IN/IN来实现这一点 多聚化、IN/宿主因子相互作用或IN/RNA相互作用。INI1/hSNF5是第一个结合内的宿主因子 以确定身份。我们已经广泛研究了它在HIV-1复制中的作用，发现它是HIV-1所必需的 晚间活动。我们发现INI1(INI1183-292)的一个最小IN结合结构域的表达被称为S6，中断 IN/INI1在体内的相互作用，并有效地抑制HIV-1颗粒的产生。推倒INI1和使用INI1-/- 细胞系也能抑制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 形态缺陷粒子的产生。这些研究共同表明，靶向IN/INI1 相互作用是抑制HIV-1颗粒产生的有效策略。然而，缺乏INI1和INI1的结构 IN/INI1的相互作用使我们无法开发针对这种相互作用的抑制剂。近期 我们实验室在解决INI1结合重复序列1(Rpt1)结构域的核磁共振结构方面的进展， 而对IN/INI1相互作用的分子对接研究有助于克服这一知识鸿沟。这些 结构研究已经通过突变、生化和病毒学研究得到验证，这些研究建立了 IN/INI1相互作用的意义。 在我们的结构研究中，我们有了一个史无前例的新发现，INI1 Rpt1和Trans HIV-1基因组RNA的激活反应元件(TAR)在结构上相互模仿。核酸拟态 By蛋白质在自然界中存在，但Rpt1模仿TAR是新的，以前还没有报道。我们发现 Rpt1和TAR结合到IN C-末端结构域(CTD)的同一表面并相互竞争 与IN结合的IC50值相同，为0.005微米。此外，IN的INI1-相互作用缺陷突变体 在颗粒形态发生障碍方面，表明这些突变体在体内不与RNA结合。《知识》 关于Rpt1和TAR之间的结构模仿为靶向这些相互作用提供了新的策略。 基于Rpt1结构域同时破坏IN/INI1和IN/TAR相互作用的事实，我们假设 从Rpt1衍生的肽类化合物具有双重活性，并抑制IN/INI1和IN/TAR相互作用。因此， 利用IN/INI1相互作用设计抑制剂具有“一举两得”的效果。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 具有增强的α螺旋性、细胞穿透特性和抗病毒力的抗病毒多肽 通过多肽装订进行蛋白质水解酶。在AIM II中，我们将测试装订多肽对IN-INI1，IN-RNA的影响 相互作用和对HIV-1复制的影响：这些研究可能会产生新的装订双活性多肽 靶向细胞内IN/INI1和/或IN/RNA相互作用以抑制HIV-1晚期事件。

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.