HTS Targeting HIV-1 Protease Autoprocessing for First in Class Drug Discovery

HTS 靶向 HIV-1 蛋白酶自动处理以实现一流药物发现

• 批准号: 10553592
• 负责人: CHAOPING CHEN
• 金额: $ 51.07万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553592
• 关键词: Affinity; Binding; Biochemical; Biological; Biological Assay; Biological Sciences; Catalogs; Catalytic Domain; Cells; Chemicals; Collaborations; Collection; Combined Modality Therapy; Drug Design; Drug resistance; Enzyme-Linked Immunosorbent Assay; Enzymes; FDA approved; Fractionation; Genetic; Goals; HIV; HIV-1; HIV-1 protease; Lead; Life; Longevity; Mediating; Medical; Michigan; Microbe; Molecular Conformation; Mutation; Natural Products; Patients; Peptide Hydrolases; Performance; Phenotype; Predisposition; Process; Production; Prognosis; Protease Inhibitor; Proteolysis; RNA-Directed DNA Polymerase; Reaction; Reporting; Resistance; Sagittaria; Structure; Technology; Therapeutic; Therapeutic Agents; Time; Treatment Efficacy; Universities; Viral; Virion; Virus Inhibitors; analog; antiretroviral therapy; assay development; combat; drug discovery; follow-up; high throughput screening; improved; inhibitor; insight; novel; novel therapeutic intervention; novel therapeutics; pol Gene Products; resistant strain; response; screening; small molecule; small molecule libraries; therapeutic development; therapeutic target; therapy outcome

HTS Targeting HIV-1 Protease Autoprocessing for First in Class Drug Discovery Project Summary This proposal is in response to PAR-17-438: Assay development and screening for discovery of chemical probes or therapeutic agents (R01). The goal of this project is to carry out high-throughput screens and follow-up characterizations to identify molecules inhibiting HIV-1 protease (PR) autoprocessing with modes of action (MOAs) different from the currently available protease inhibitors (PIs). In the infected cell, HIV-1 PR is initially synthesized as part of the Gag-Pol polyprotein precursor with its proteolysis activity tightly suppressed. During late stage of virion production, the precursor self-catalyzes the cleavage reactions that lead to liberation of the free mature PR in a temporospatially regulated fashion. The FDA-approved HIV-1 PIs primarily target the catalytic site of the mature PR and they are significantly less effective at suppressing precursor-mediated autoprocessing, suggesting that these two forms of HIV-1 PR are enzymatically different. Also, the emergence of PI resistant strain in patients treated with PI-containing combination antiretroviral therapy (cART) is an ongoing problem that diminishes treatment efficacy, which warrants the need for new therapeutics. This project seeks to find novel autoprocessing inhibitors targeting the precursor at regions not recognized by the currently available PIs. Towards this goal, we have established a cell-based functional assay that has faithfully recapitulated the autoprocessing phenotypes observed with proviral constructs. This assay has also, for the first time, made it possible to screen for autoprocessing inhibitors by HTS using AlphaLISA (amplified luminescent proximity homogeneous assay ELISA) technology. Our pilot screens of ~26K small molecule compounds displayed satisfactory performance with Z’ factors >0.45, S/N ratios >10, and hit rates <0.1% although no confirmed hit was identified. Therefore, we plan to screen a collection of natural product extracts (40K extracts, 5-25 compounds per extract, totaling ~0.6 million chemicals) with a wild type and two PI resistant precursors in collaboration with Dr. David Sherman at University of Michigan Life Sciences Institute (Aim 1). In parallel, we will team up with Drs. Thomas Chung and Ian Pass at Sanford Burnham Prebys Medical Discovery Institute to screen their ~350K small molecule library (Aim 2). These HTS campaigns will hopefully identify a handful confirmed compounds that will be subjected to a battery of established secondary and tertiary assays (Aim 3) in order to find novel autoprocessing inhibitors that are different from the current HIV-1 PIs in their MOA. This next generation of therapeutic probes, when used in combination with the current PIs, will implement a new therapeutic approach: targeting a vital enzyme (HIV-1 protease) at two distinct functional states (precursor and mature PR) and at different regions (non-catalytic and catalytic sites) at the same time. Such a strategy is expected to drastically increase difficulty (genetic barrier) for HIV-1 to evolve viable strains simultaneously resistant to inhibitors from both classes to resist the resistance.

HTS靶向HIV-1蛋白酶在类药物发现中首先自动化 项目摘要 该提案是对PAR-17-438的回应：发现化学问题的测定开发和筛选 或治疗剂（R01）。该项目的目的是执行高通量屏幕和后续屏幕 鉴定抑制HIV-1蛋白酶（PR）自动化的分子的表征 （MOA）与当前可用的蛋白酶抑制剂（PI）不同。在受感染的细胞中，HIV-1 PR最初是 合成为GAG-POL多蛋白前体的一部分，其蛋白水解活性严重抑制。期间 病毒粒子生产的后期，前体的自我催化会导致解放的裂解反应 以临时受监管的方式自由成熟的公关。 FDA批准的HIV-1 PIS主要目标 成熟PR的催化位点，它们在抑制前体介导的效果明显较小 自动加入，表明这两种形式的HIV-1 PR在酶上是不同的。另外，出现 用含PI组合抗逆转录病毒治疗（CAR）治疗的患者的PI抗性菌株是一种正在进行的 降低治疗效率的问题，需要新的治疗剂。这个项目试图 查找针对前体的新型自动加入抑制剂，该抑制剂在目前可用的区域未识别的区域 pis。为了实现这一目标，我们建立了一个基于细胞的功能测定法，该测定法已忠实地概括了 用临时构建体观察到的自动加入表型。该测定也是第一次 可以使用alphalisa筛选HTS自动加入抑制剂（放大发光接近度 均质测定ELISA）技术。我们显示的〜26K小分子化合物的试验屏幕显示 Z'> 0.45，S/N比> 10，命中率<0.1％，令人满意的性能令人满意 因此，我们计划筛选自然产品提取物的集合（40k提取物，5-25 每提取物的化合物，总计约60万种化学物质），具有野生型和两个抗PI的前体 与密歇根大学生命科学学院的David Sherman博士合作（AIM 1）。并行，我们 将与Drs合作。 Sanford Burnham Prebys医学发现研究所的Thomas Chung和Ian Pass到 筛选其〜350K小分子库（AIM 2）。这些HTS运动有望确定少数 确认的化合物将受到一系列已建立的二级和第三次测定（AIM 3） 为了找到与MOA中当前HIV-1 PIS不同的新型自动化抑制剂。接下来 当与当前PI结合使用时，生成治疗问题将实施新的 治疗方法：针对两个不同功能状态的重要酶（HIV-1蛋白酶）（前体和 同时在不同区域（非催化和催化位点）的成熟PR）。这样的策略是 预计HIV-1简单地进化可行菌株会大大增加困难（遗传障碍） 两种类别的抑制剂具有抵抗力的抵抗力。