Integrase Binding Proteins as Drug Targets to Inhibit HIV-1 Assembly

描述：目前还没有FDA批准的针对病毒组装的药物来对抗艾滋病。我们的长期目标是开发一类新的药物，针对Gag-Pol来破坏整合酶(IN)-宿主蛋白的相互作用，以抑制HIV组装的早期阶段。在HIV-1晚期期间 在这种情况下，新生病毒蛋白通过细胞质运输到质膜进行组装和发芽。GAG是体外组装的必要条件和充分条件。然而，遗传学研究表明，整合酶(IN)区域的突变也会导致颗粒形态、病毒粒子释放以及后续感染事件中的缺陷。此外，最近的工作表明，IN的变构抑制剂抑制了颗粒的形态形成和感染性。我们已经证明了IN与宿主因子整合酶相互作用因子1(INI1)/hSNF5结合缺陷的突变体能够有效地抑制颗粒的形态发生，表明通过破坏整合酶来抑制组装是一个可行的策略。INI1/hSNF5是SWI/SNF染色质重塑复合体的一个组成部分，与IN结合。我们的研究表明INI1在HIV-1组装中起着至关重要的作用。干扰INI1功能通过阻断Gag/Gag-Pol的运输和稳定性而有效地抑制(1000倍)组装。INI1是一种适配蛋白，我们假设它在组装中间体与必要的细胞成分之间的相互作用之间架起了桥梁，以促进Gag/GagPol向质膜的正确运输。新的见解揭示，影响REV依赖的HIV-1RNA输出的蛋白质与INI1相关，将组装与RNA输出联系起来。我们认为，针对IN-INI1相互作用的策略将导致抑制独特的早期组装步骤，即GAG贩运和稳定性。在目标I中，我们将研究INI1促进HIV-1组装的机制以及INI1突变如何破坏这一过程。我们将确定干扰INI1是否同时抑制RNA出口和GAG贩运。在AIM II中，我们将确定HIV-1复制如何影响INI1核输出，因为INI1的这一功能似乎是中介组装所必需的。在目标III中，我们将把我们的观察转化为开发一类针对IN-INI1相互作用的新型药物，以抑制GAG细胞质转运。我们将进行基于分子动力学(MD)同源性的计算建模，以预测最小IN结合结构域(S6)的结构，根据结构产生多肽模拟物，建立基于AlphaScreen的高通量筛选(HTS)分析，并筛选干扰IN-INI1相互作用的化合物。

DESCRIPTION: Currently there are no FDA approved drugs targeting viral assembly to combat AIDS. Our long-term goal is to develop a new class of drugs that target Gag-Pol to disrupt integrase (IN)-host protein interactions to inhibit early stages of HIV assembly. During HIV-1 late events, the nascent viral proteins traffic through the cytoplasm to the plasma membrane for assembly and budding. Gag is necessary and sufficient for assembly in vitro. However, genetic studies reveal that mutations in the Integrase (IN) region also leads to defects in particle morphology, virion release, as well as defect in subsequent events of infection. Furthermore, recent work demonstrates that allosteric inhibitors of IN inhibit particle morphogenesis and infectivity. We have shown that mutants of IN defective for binding to a host factor Integrase interactor 1 (INI1)/hSNF5 potently inhibit particle morphogenesis, indicating that disrupting integrase to inhibit assembly is a feasible strategy. INI1/hSNF5 is a component of the SWI/SNF chromatin-remodeling complex that binds to IN. Our studies indicate an essential role for INI1 in HIV-1 assembly. Interfering with INI1 function potently inhibits (>1000 fold) assembly by blocking Gag/Gag-Pol trafficking and stability. INI1 is an adaptor protein and we hypothesize that it bridges interaction of assembly intermediates with essential cellular components to facilitate proper trafficking of the Gag/GagPol to the plasma membrane. New insights reveal that proteins that influence Rev-dependent HIV-1 RNA export are associated with INI1, linking assembly to RNA export. We propose that strategies to target IN-INI1 interactions will lead to inhibition of unique and early steps of assembly namely, Gag trafficking and stability. In Aim I, we will investigate the mechanism by which INI1 facilitate HIV-1 assembly and how INI1 mutants disrupt this process. We will determine if interfering with INI1 inhibits both RNA export and Gag trafficking. In Aim II, we will determine how HIV-1 replication influences INI1 nuclear export, as this function of INI1 appears is necessary for mediating assembly. In Aim III, we will translate our observations into developing a novel class of drugs targeting IN-INI1 interactions to inhibit Gag cytoplasmic trafficking. We will carry out molecular dynamics (MD) homology-based computational modeling to predict the structure of minimal IN binding domain (S6), generate peptidomimetics based on the structure, establish high throughput screening (HTS) assays based on AlphaScreen, and screen for compounds to disrupt IN-INI1 interaction.