Targeting Tat/TAR interactions with the superelongation complex to develop novel treatments for HIV/AIDS

靶向 Tat/TAR 与超伸长复合物的相互作用，开发艾滋病毒/艾滋病的新疗法

• 批准号: 10304202
• 负责人: URSULA SCHULZE-GAHMEN
• 金额: $ 23.63万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-17 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10304202
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Adherence; Affinity; Binding; Biochemistry; Biological Assay; California; Complement Factor B; Complex; Coupled; Crystallography; Cyclin-Dependent Kinases; Development; Drug Design; Drug Interactions; Elongation Factor; Enzymes; Event; Feedback; Fluorescence Polarization; Funding; Genetic Transcription; Goals; Grant; HIV; HIV-1; Human; Hydrophobicity; Incidence; Infection; Integrase; Knowledge; Label; Laboratories; Life; Ligand Binding; Ligands; Malignant Neoplasms; Mission; Nature; Patients; Peptide Hydrolases; Peptides; Persons; Pharmaceutical Preparations; Positive Transcriptional Elongation Factor B; Protein Region; Proteins; Public Health; RNA-Directed DNA Polymerase; Research; Research Design; Research Support; Resistance; Roentgen Rays; San Francisco; Scientist; Structure; Surface; Testing; Therapeutic; Ubiquitination; United States National Institutes of Health; Universities; Viral; Viral Proteins; Virus Replication; Work; X-Ray Crystallography; antiretroviral therapy; base; cyclin T1; dosage; drug structure; expectation; experience; high throughput screening; high-throughput drug screening; inhibitor; method development; neglect; new therapeutic target; novel; novel strategies; novel therapeutic intervention; novel therapeutics; promoter; protein complex; protein degradation; recruit; response; screening; small molecule; small molecule inhibitor; small molecule libraries; structural biology; success; targeted treatment; three dimensional structure; tool; transcription factor; ubiquitin-protein ligase; virtual screening

PROJECT SUMMARY (ABSTRACT) Despite the remarkable success of antiretroviral therapy against human immunodeficiency virus 1 (HIV-1), novel therapeutics are needed to address issues of resistance, tolerability, drug-drug interactions, and variable adherence to daily drug regiments. The objective of this application is to develop novel therapeutic strategies to expand the tools for HIV-treatment. The central hypothesis is that the interactions of TAR with the superelongation complex (SEC) can be inhibited by small-molecule drugs, and binding pockets on the Tat/Cyclin T surface close to TAR can be targeted by structure-based virtual screening for developing PROTAC molecules. The central hypothesis will be tested in a two-pronged approach. Aim 1: To identify and validate small-molecule inhibitors of TAR binding by high-throughput screening. After optimizing the fluorescence polarization binding assay for TAR binding to the SEC, we will extensively screen libraries from the Small Molecule Discovery Center at UCSF and characterize identified inhibitors functionally and structurally with transcription assays, crystallography to further optimize the ligands, and latency reversal assays. Preliminary results show the feasibility of a high-throughput FP assay with labelled TAR and purified SEC. We expect to identify several inhibitors of TAR binding by the end of the grant period. Aim 2: To identify Tat-specific ligands to Tat-AFF4-SEC with the goal of developing chimeric molecules that target Tat complexes for degradation (PROTACs). We identified several pockets that are mostly or partially defined by Tat residues. We will perform structure-based virtual screening (SBVS) to identify small molecule candidates for Tat-dependent binding to the Tat SEC. SBVS will be executed with the help of the Shoichet laboratory (UCSF). Top candidates will be tested in TAR binding assays with the expectation that ligand binding to surface pockets adjacent to Tat and TAR will inhibit TAR binding. We will determine the structures of ligand binding complexes by X-ray crystallography to guide further drug design studies. Such ligands will be a first step towards creating chimeric E3-ligase recruiting PROTACs. PROTACs offer a novel approach with the advantage of theoretically requiring lower affinity transient binding events that are of catalytic nature, avoiding high-level drug dosages. By targeting new HIV complexes with novel drug development methods, we intend to expand the repertoire of effective HIV drugs. The proposed research is expected to contribute to the development of novel therapeutics, targeting so-far neglected intracellular viral proteins and expanding druggability to additional intracellular HIV proteins and their complexes with host proteins. We expect our contributions will significantly advance the field as they are aimed at establishing robust screening assays for a novel HIV-1 target that regulates viral replication.

项目概要（摘要） 尽管针对人类免疫缺陷病毒1（HIV-1）的抗逆转录病毒疗法取得了显着的成功，但新的抗逆转录病毒疗法仍然存在。 需要治疗来解决耐药性、耐受性、药物间相互作用和可变性等问题。 坚持每日用药方案。本申请的目的是开发新的治疗策略， 扩大艾滋病毒治疗的工具。中心假设是，TAR与 超伸长复合物（SEC）可被小分子药物抑制，达特/细胞周期蛋白上的结合口袋 可以通过基于结构的虚拟筛选靶向靠近TAR的T表面以开发PROTAC分子。 中心假设将以双管齐下的方法进行检验。目的1：鉴定和验证小分子 通过高通量筛选筛选TAR结合抑制剂。优化荧光偏振结合后 为了检测TAR与SEC的结合，我们将广泛筛选来自小分子发现中心的文库 在加州大学旧金山分校和表征识别抑制剂的功能和结构与转录测定， 晶体学以进一步优化配体和潜伏期逆转测定。初步结果显示， 使用标记的TAR和纯化的SEC的高通量FP测定的可行性。我们希望能找到几个 在授权期结束前，TAR结合抑制剂。目的2：鉴定Tat-AFF 4-SEC的Tat特异性配体 目的是开发靶向达特复合物降解的嵌合分子（PROTAC）。我们 鉴定了几个大部分或部分由达特残基限定的口袋。我们将执行基于结构的 虚拟筛选（SBVS）以鉴定Tat依赖性结合至达特SEC的小分子候选物。SBVS 将在Shoichet实验室（UCSF）的帮助下执行。最佳候选人将在TAR绑定测试 预期配体与邻近达特和TAR的表面口袋结合将抑制TAR的测定 约束力我们将通过X射线晶体学确定配体结合复合物的结构，以指导进一步的研究。 药物设计研究。这样的配体将是产生嵌合E3-连接酶募集PROTAC的第一步。 PROTAC提供了一种新的方法，其优点是理论上需要较低的亲和力瞬时结合 具有催化性质的事件，避免高水平的药物剂量。通过靶向新的HIV复合物， 在药物开发方法方面，我们打算扩大有效的艾滋病毒药物库。拟议研究 有望有助于开发新的治疗方法，靶向迄今被忽视的细胞内病毒 蛋白质和扩大对其他细胞内HIV蛋白质及其与宿主的复合物的可药性 proteins.我们希望我们的贡献将大大推进该领域，因为它们旨在建立强大的 一种新的HIV-1靶点的筛选试验，该靶点可调节病毒复制。