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靶向Gag-Pol的新型药物，以破坏整合酶（IN）-宿主蛋白相互作用，从而抑制HIV组装的早期阶段。在HIV-1晚期 在这些事件中，新生的病毒蛋白质通过细胞质运输到质膜进行组装和出芽。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缺陷、病毒体释放以及后续感染事件的缺陷。此外，最近的工作表明，变构抑制剂的IN抑制颗粒形态和感染性。我们已经表明，突变体的IN缺陷的结合宿主因子整合酶相互作用1（INI 1）/hSNF 5有效地抑制颗粒形态发生，这表明，破坏整合酶，以抑制组装是一个可行的策略。 INI 1/hSNF 5是结合IN的SWI/SNF染色质重塑复合物的组分。我们的研究表明INI 1在HIV-1组装中起重要作用。干扰INI 1功能通过阻断Gag/Gag-Pol运输和稳定性而有效抑制（>1000倍）组装。INI 1是一种衔接蛋白，我们假设它桥接组装中间体与必需细胞组分的相互作用，以促进Gag/GagPol向质膜的适当运输。新的见解表明，影响Rev依赖的HIV-1 RNA输出的蛋白质与INI 1相关，将装配与RNA输出联系起来。 我们建议，针对IN-INI 1相互作用的策略将导致抑制组装的独特和早期步骤，即Gag贩运和稳定性。在目的I中，我们将研究INI 1促进HIV-1组装的机制以及INI 1突变体如何破坏这一过程。我们将确定干扰INI 1是否抑制RNA输出和Gag运输。在目的II中，我们将确定HIV-1复制如何影响INI 1核输出，因为INI 1的这种功能似乎是介导组装所必需的。在目标III中，我们将把我们的观察转化为开发一类新的药物，靶向IN-INI 1相互作用，以抑制Gag细胞质运输。我们将进行基于分子动力学（MD）同源性的计算建模，以预测最小IN结合结构域（S6）的结构，基于该结构生成肽模拟物，建立基于AlphaScreen的高通量筛选（HTS）测定，并筛选破坏IN-INI 1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.