RNA-mimicry to guide the intra-cellular targeting of host virus protein and viral RNA-protein interactions to inhibit HIV replication.

摘要： 本申请长期目标是将宿主-病毒相互作用界面表征为一种新的药物靶点 开发抑制剂来干扰宿主和病毒之间的细胞内蛋白质-蛋白质相互作用(PPI)以 遏制HIV-1复制。已经证实，在不影响其酶活性的情况下干扰它可以 抑制HIV-1复制的后期阶段，如组装、颗粒生产和/或颗粒形态形成。 这种II类IN突变和IN(Allini)的变构抑制剂抑制晚期事件，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，这些缺陷在 在与宿主因子相互作用缺陷的IN突变体中也可以观察到颗粒形态发生， INI1/hSNF5，一种IN结合的宿主因子，选择性地整合到HIV-1病毒粒子中。HIV需要INI1- 1迟来的事件。INI1最小IN结合区(INI1183-292)S6的表达，破坏IN/INI1 在体内相互作用，并有效地抑制HIV-1颗粒的产生。INI1基因敲除及INI1-/-细胞系的应用 抑制HIV-1颗粒的产生。突变体中的InI1结合缺陷导致形态上的 缺陷颗粒提示靶向IN/INI1相互作用是抑制HIV-1颗粒的有效策略 制作。缺乏INI1和IN/INI1复合体的结构使我们无法开发出靶向的抑制剂 这种互动。我们实验室的新研究，包括IN-结合重复序列1(Rpt1)的核磁共振结构 INI1的结构域以及IN/INI1相互作用的分子对接有助于克服这一知识鸿沟。 我们发现INI1的IN结合域，称为Rpt1，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。Rpt1和TAR结合在同一表面 并以相同的IC50值(0.005微米)竞争与IN的结合。 此外，IN的INI1-相互作用缺陷突变导致颗粒形态发生障碍。我们 假设多肽类药物和源自Rpt1的小分子具有双重活性并同时抑制两者 In/INI1和IN/TAR相互作用。作为原理的证明，我们已经开发出一种从 Rpt1的a-1螺旋界面，有效地破坏IN/INI1和IN/RNA相互作用，抑制颗粒形态形成 和体内HIV-1复制。在这项提案中，我们将把IN/INI1界面描述为一个杰出的药物靶点 通过进行：i)遗传分析，以了解INI1对集合体/粒子的影响机制 通过反式互补和“合成救援”实验生产；ii)开发一类新的 具有双重靶向IN/INI1和IN/RNA相互作用的单链多肽和小分子 虚拟配体的筛选及含IN-C-末端的INI-1-钉合肽配合物的核磁共振结构测定 结构域；以及iii)了解INI1-衍生的装订多肽和小分子的机制 通过筛选病毒逃逸突变体抑制HIV-1复制和靶标识别。这些研究将 建立IN/INI1作为新的药物靶点，并提供新的先导化合物来抑制HIV-1晚期事件。

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.