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

描述：目前 FDA 还没有批准针对病毒组装来对抗艾滋病的药物。我们的长期目标是开发一类新型药物，以 Gag-Pol 为靶点，破坏整合酶 (IN) 与宿主蛋白的相互作用，从而抑制 HIV 组装的早期阶段。 HIV-1晚期期间 事件发生后，新生病毒蛋白通过细胞质运输到质膜进行组装和出芽。 Gag 对于体外组装来说是必要且充分的。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、病毒颗粒释放的缺陷以及后续感染事件的缺陷。此外，最近的工作表明 IN 的变构抑制剂抑制颗粒形态发生和感染性。我们已经证明，与宿主因子整合酶相互作用蛋白 1 (INI1)/hSNF5 结合缺陷的 IN 突变体可有效抑制颗粒形态发生，表明破坏整合酶以抑制组装是一种可行的策略。 INI1/hSNF5 是与 IN 结合的 SWI/SNF 染色质重塑复合物的组成部分。我们的研究表明 INI1 在 HIV-1 组装中发挥着重要作用。干扰 INI1 功能可通过阻断 Gag/Gag-Pol 运输和稳定性来有效抑制（>1000 倍）组装。 INI1 是一种衔接蛋白，我们假设它桥接了组装中间体与必需细胞成分的相互作用，以促进 Gag/GagPol 正确运输至质膜。新的见解表明，影响 Rev 依赖性 HIV-1 RNA 输出的蛋白质与 INI1 相关，将组装与 RNA 输出联系起来。 我们建议针对 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 运输和稳定性。在目标 I 中，我们将研究 INI1 促进 HIV-1 组装的机制以及 INI1 突变体如何破坏这一过程。我们将确定干扰 INI1 是否会抑制 RNA 输出和 Gag 运输。在目标 II 中，我们将确定 HIV-1 复制如何影响 INI1 核输出，因为 INI1 的这一功能似乎对于介导组装是必要的。在目标 III 中，我们将把我们的观察结果转化为开发一类针对 IN-INI1 相互作用的新型药物，以抑制 Gag 细胞质运输。我们将进行基于分子动力学（MD）同源性的计算模型来预测最小IN结合域（S6）的结构，根据该结构生成肽模拟物，建立基于AlphaScreen的高通量筛选（HTS）测定法，并筛选破坏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.