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

项目总结 艾滋病毒感染仍然是一个重大的世界健康问题，需要新的方法来治疗艾滋病。整合酶(IN)是一种病毒- 一种编码的酶，对逆转录病毒的复制至关重要，是发展的既定目标 治疗艾滋病毒/艾滋病的药物。五种FDA批准的针对活性部位的药物正在临床上用于治疗艾滋病 患者，但交叉耐药病毒变种已经被很好地记录下来。抗击对现有IN的抵抗 活性部位抑制剂，我们需要利用针对其他所需病毒蛋白的作用机制的药物 功能，如多聚体或与病毒和细胞共因子的相互作用。一种很有前途的新方法是 集中在开发针对非活性部位(或变构)位置的抑制剂，但这些都没有 已批准临床使用。 为了发挥正常的功能，整合酶必须形成能够执行两种病毒的协同插入的多聚体 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可能性 多聚体作为一种有效的抗病毒策略。除了表征良好的催化核心二聚体 界面，我们的研究揭示了二聚体界面是由N-末端和 催化核心区，这也观察到在最近的冷冻-EM结构的HIV内质粒。建议数 研究以这种新的蛋白质-蛋白质相互作用为目标，并建议对破坏这种相互作用的化合物进行表征 IN活动需要接口。 作为一种识别变构抑制剂的唯一方法，它针对的是蛋白质-蛋白质相互作用 功能整合酶多聚体，我们设计了一种基于FRET的方法，专门检测 N-末端结构域-催化核心结构域二聚体。在这项试验的第一个版本中，我们鉴定了几种化合物 这也抑制了IN的催化活性，更重要的是，其中两种化合物抑制了HIV对细胞的感染 在文化上。然而，毒性方面的担忧限制了这些早期药物的实用性。我们已经开发出一种改进的， 第二代筛选，并从专门的化学库中鉴定了25种新的热门化合物 旨在针对蛋白质之间的相互作用。这些新的候选化合物中有8个被认为 可接受严格的类药物质量标准，并具有很强的药物化学潜力 进步。拟议工作的目的是：目标1-验证这些HIT化合物，包括详细的 生物化学特性；以及目标2-测试化合物的细胞毒性和阻断病毒感染的能力。 从这一应用程序的实验中获得的信息将对 联合使用有效的HIV变构抑制剂的抗艾滋病药物的开发 用现有的药物来防止出现耐药病毒株。

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.