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.

项目概要 HIV 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。编码的酶对于逆转录病毒复制至关重要，并且是开发的既定目标治疗艾滋病毒/艾滋病的药物有五种 FDA 批准的针对活性位点的药物正在临床用于治疗艾滋病。患者，但交叉耐药病毒变种已被充分记录，以对抗现有 IN 的耐药性。活性位点抑制剂，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，例如多聚化或与病毒和细胞辅助因子的相互作用是一种有前途的新方法。集中于开发针对非活性位点（或“变构”）位置的抑制剂，但这些都没有被批准用于临床。为了发挥正常功能，整合酶必须形成多聚体，能够协调插入两个病毒 DNA 末端进入宿主目标 DNA，称为协同整合，我们将测试适当抑制的潜力。除了充分表征的催化核心二聚体之外，IN 多聚化也是一种有效的抗病毒策略。界面，我们的研究揭示了由 N 端和 N 端之间的相互作用介导的二聚体界面催化核心结构域，在最近的 HIV 嵌体冷冻电镜结构中也观察到了这一点。研究针对这种新颖的蛋白质-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。作为识别变构抑制剂的独特方法，该抑制剂针对蛋白质-蛋白质相互作用所需的为了实现功能性整合酶多聚化，我们设计了一种基于 FRET 的检测方法，专门检测 N 末端结构域-催化核心结构域二聚体在该测定的第一个版本中，我们鉴定了几种化合物。还抑制 IN 催化活性，重要的是其中两种化合物抑制 HIV 细胞感染然而，毒性问题限制了这些早期热门产品的实用性。第二代筛选，并从专业化学库中鉴定出 25 种新的热门化合物旨在针对蛋白质-蛋白质相互作用的这些新化合物中的八种被认为是。符合严格的类药质量标准，并且具有强大的药物化学潜力拟议工作的目的是：目标 1 - 验证这些热门化合物，包括详细信息。生化表征；目标 2 - 测试化合物的细胞毒性和阻止病毒感染的能力。从本应用中的实验中获得的信息将有可能产生重大影响开发抗艾滋病药物，作为有效的 HIV IN 变构抑制剂，联合使用与现有药物一起预防耐药病毒株的出现。