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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。

基本信息

批准号：
10618961
负责人：
GANJAM V KALPANA
金额：
$ 67.7万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
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

项目摘要

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。