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

破坏 HIV-1 整合酶二聚化的新型变构抑制剂的表征

基本信息

批准号：
10308105
负责人：
MARK David ANDRAKE
金额：
$ 23.38万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10308105
关键词：
AIDS/HIV problem Acquired Immunodeficiency Syndrome Active Sites Affinity Anti-HIV Agents Architecture Binding Biochemical Biological Biological Assay Biophysics Catalysis Catalytic Domain Cells Chemicals Clinical Cryoelectron Microscopy DNA Development Dimerization Dose Drug Targeting Drug Utilization Enzymes Event FDA approved Fluorescence Resonance Energy Transfer Generations Goals HIV HIV Infections HIV Integrase HIV Integrase Inhibitors HIV-1 integrase In Vitro Integrase Integrase Inhibitors Life Cycle Stages Location Measures Mediating Methods N-terminal Patients Pharmaceutical Chemistry Pharmaceutical Preparations Proteins Resistance Roentgen Rays Structural Models Structure Targeted Research Testing Toxic effect Variant Viral Viral Genome Viral Proteins Virus Virus Diseases Virus Integration Work World Health base biophysical analysis combat crosslink design dimer drug development drug standard experimental study improved inhibitor novel novel strategies particle prevent protein function protein protein interaction reproductive response small molecule libraries viral DNA

项目摘要

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.

项目摘要艾滋病毒感染仍然是一个主要的世界卫生问题，需要治疗艾滋病的新方法。整合酶（in）是一种病毒 - 对于逆转录病毒复制至关重要的编码酶，是开发的既定目标治疗艾滋病毒/艾滋病的药物。针对活动部位的五种FDA批准的药物用于治疗艾滋病患者，但越野抗性的病毒变体已经有充分的文献证明。打击对现有的抵抗活性位点抑制剂，我们需要使用针对其他必需病毒蛋白的作用机制的药物功能，例如多层化或与病毒和细胞共同因素的相互作用。一个有希望的新方法是集中于开发针对非活动部位（或“变构”）位置的抑制剂，但这些都不是批准用于临床用途。为了适当的功能，集成酶必须形成有能力执行两个病毒的协调插入的多IMR DNA终止于宿主目标DNA，称为协同集成。我们将测试抑制适当的潜力作为一种有效的抗病毒策略，在多封件中。除了特征良好的催化核心二聚体界面，我们的研究揭示了由N末端与催化核心结构域，这也可以在HIV肠的最近的冷冻EM结构中观察到。提议研究以这种新型的蛋白质 - 蛋白质相互作用和建议来表征破坏这一点的化合物活动所需的接口。作为识别针对蛋白质 - 蛋白质相互作用所需的蛋白质蛋白质相互作用的独特方法功能集成的多层化，我们设计了一个基于FRET的测定法，该测定法专门检测 N末端域催化核心结构域二聚体。在此测定的第一个版本中，我们确定了几种化合物这也抑制了催化活性，重要的是，其中两种抑制了细胞的HIV感染在文化中。但是，毒性涉及这些早期流行的有用性。我们已经开发了一个改进的第二代屏幕，并从专业化学库中确定了25种新的命中化合物旨在靶向蛋白质 - 蛋白质相互作用。这些新候选化合物中有八种被认为是可以通过严格的类似药物的品质和潜在的药物化学潜力接受进步。拟议工作的目的是：目标1-验证这些命中化合物，包括详细的生化特征； AIM 2-针对细胞毒性的测试化合物和阻断病毒感染的能力。从本申请中的实验中获得的信息将有可能对开发抗AID的药物，可作为HIV的有效变构抑制剂，用于组合使用现有药物来防止抗性病毒菌株的出现。