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

项目摘要(摘要) 尽管针对人类免疫缺陷病毒1型(HIV-1)的抗逆转录病毒疗法取得了显著的成功，但新的 需要治疗学来解决耐药性、耐受性、药物-药物相互作用和变量 坚持每天服药。这项应用的目标是开发新的治疗策略，以 扩大艾滋病毒治疗的工具。中心假设是焦油与石油的相互作用 超延长复合体(SEC)可被小分子药物和TAT/Cyclin上的结合口袋所抑制 接近焦油的T表面可以通过基于结构的虚拟筛选来开发PROTAC分子。 核心假设将通过双管齐下的方法进行检验。目标1：鉴定和验证小分子 高通量筛选焦油结合抑制剂。优化后的荧光偏振结合 为了检测TAR与SEC的结合，我们将从小分子发现中心广泛筛选文库 在加州大学旧金山分校，并用转录试验从功能和结构上表征鉴定的抑制剂， 结晶学，进一步优化配基，并进行潜伏期逆转分析。初步结果显示， 用标记的焦油和纯化的SEC进行高通量FP检测的可行性。我们预计将确定几个 在授权期结束前使用焦油结合抑制剂。目的2：确定TAT-AFF4-SEC的TAT特异性配体 目标是开发针对TAT络合物进行降解的嵌合分子(PROTAC)。我们 确定了几个主要或部分由TAT残留物定义的口袋。我们将执行基于结构的 虚拟筛选(SBVS)，以确定依赖于TAT与TAT SEC结合的小分子候选。SBVS 将在Shoichet实验室(UCSF)的帮助下执行。顶尖候选人将接受焦油绑定测试 预计与TAT和TAR相邻的表面口袋上的配体结合将抑制TAR 有约束力的。我们将通过X射线结晶学确定配体结合络合物的结构，以指导进一步 药物设计研究。这种配体将是创建嵌合E3-连接酶招募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亲和力的瞬时结合 具有催化性质的事件，避免高水平的药物剂量。通过以新型艾滋病毒复合体为靶点 在药物开发方法方面，我们打算扩大有效的艾滋病毒药物的保留库。拟议的研究 有望为开发针对迄今被忽视的细胞内病毒的新疗法做出贡献 蛋白质及其对细胞内额外HIV蛋白及其与宿主的复合体的可药性扩展 蛋白质。我们希望我们的贡献将大大推动这一领域的发展，因为它们的目标是建立强大的 筛选一种调节病毒复制的新的HIV-1靶点。

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.