Characterization of New Allosteric Inhibitors that Disrupt HIV-1 Integrase Dimerization

项目摘要 艾滋病毒感染仍然是一个主要的世界卫生问题，需要新的方法来治疗艾滋病。整合酶（IN）是一种病毒- 编码的酶，是逆转录病毒复制所必需的，是发展的既定目标， 治疗艾滋病的药物。FDA批准的五种针对活性位点的药物已在临床上用于治疗艾滋病 患者，但交叉耐药病毒变种已被充分记录。为了克服现有IN的阻力， 活性位点抑制剂，我们需要利用靶向其他所需病毒蛋白的作用机制的药物 功能，如多聚化或与病毒和细胞辅因子的相互作用。一种有前途的新方法是 集中于开发靶向非活性位点（或“变构”）位置的抑制剂，但这些都没有被 批准用于临床。 为了正常发挥功能，整合酶必须形成多聚体，以便协调插入两个病毒载体。 DNA末端进入宿主靶DNA，称为协同整合。我们将测试抑制适当的 IN多聚化作为有效的抗病毒策略。除了充分表征的催化核心二聚体之外， 界面，我们的研究揭示了二聚体界面介导的相互作用之间的N-末端和 催化核心结构域，这也在最近的HIV整合体的冷冻-EM结构中观察到。拟议 研究的目标是这种新的蛋白质-蛋白质相互作用，并提出表征破坏这种相互作用的化合物。 IN活动所需的接口。 作为一种独特的方法，以确定变构抑制剂的目标蛋白质-蛋白质相互作用所需的 功能性整合酶多聚化，我们设计了一种基于FRET的检测方法， N-末端结构域-催化核心结构域二聚体。在第一次试验中，我们鉴定了几种化合物， 这也抑制了IN催化活性，重要的是，这些化合物中的两种抑制了HIV感染细胞 在文化中。然而，毒性问题限制了这些早期命中的有用性。我们开发了一种改进的， 第二代筛选，并确定了25个新的命中化合物从专门的化学图书馆 旨在靶向蛋白质间的相互作用。这些新的候选化合物中有八种被认为是 通过严格的药物质量标准可接受，并具有强大的药物化学潜力， 进步。所提出的工作的目的是：目标1 -确定这些命中化合物，包括详细的 目的2 -测试化合物的细胞毒性和阻断病毒感染的能力。 从该应用程序的实验中获得的信息将有可能对以下方面产生重大影响： 开发作为HIV IN的有效变构抑制剂的抗艾滋病药物，用于组合 与现有的药物，以防止耐药病毒株的出现。

PROJECT SUMMARY HIV infection remains a major world health issue requiring new methods to treat AIDS. Integrase (IN) is a virus- encoded enzyme that is essential for retroviral replication and is an established target for the development of drugs to treat HIV/AIDS. Five FDA-approved drugs that target the active site are in clinical use to treat AIDS patients, but cross-resistant virus variants have been well documented. To combat resistance to existing IN active site inhibitors, we need drugs that utilize a mechanism of action targeting other required viral protein functions, such as multimerization or interaction with viral and cellular co-factors. A promising new approach is centered on developing inhibitors targeting non-active site (or “allosteric”) locations, but none of these have been approved for clinical use. For proper function, integrase must form multimers competent to perform the coordinated insertion of two viral DNA ends into the host target DNA, called concerted integration. We will test the potential for inhibiting proper IN multimerization as an effective antiviral strategy. In addition to the well-characterized catalytic core dimer interface, our studies have revealed a dimer interface mediated by interactions between the N-terminal and catalytic core domains, which is also observed in the recent cryo-EM structure of HIV intasomes. The proposed research targets this novel protein-protein interaction and proposes to characterize compounds that disrupt this interface required for IN activity. As a unique approach to identify allosteric inhibitors that target the protein-protein interactions required for functional integrase multimerization, we designed a FRET-based assay that specifically detects formation of the N-terminal domain-catalytic core domain dimer. In the first version of this assay, we identified several compounds that also inhibited IN catalytic activities, and importantly two of these compounds inhibited HIV infection of cells in culture. However, toxicity concerns limited the usefulness of these early hits. We have developed an improved, second-generation screen and have identified 25 new hit compounds from specialized chemical libraries designed to target protein-protein interactions. Eight of these new candidate compounds were deemed acceptable by strict standards of drug-like qualities and possess strong medicinal chemistry potential for advancement. The purpose of the proposed work is to: Aim 1 - Validate these hit compounds including a detailed biochemical characterization; and Aim 2 - Test compounds for cell toxicity and the ability to block virus infection. The information gained from the experiments in this application will have the potential for major impact in development of anti-AIDS drugs that function as effective allosteric inhibitors of HIV IN, for use in combination with existing drugs to prevent the emergence of resistant virus strains.