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 蛋白酶自动处理以实现一流药物发现 项目概要 该提案是对 PAR-17-438 的回应：化学探针发现的测定开发和筛选 或治疗剂(R01)。该项目的目标是进行高通量筛选和后续研究 鉴定抑制 HIV-1 蛋白酶 (PR) 自动加工的分子及其作用模式的表征 （MOA）与目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 作为 Gag-Pol 多蛋白前体的一部分合成，其蛋白水解活性受到严格抑制。期间 在病毒颗粒产生的后期，前体自催化裂解反应，导致病毒颗粒的释放 以时空调节的方式释放成熟的公关。 FDA 批准的 HIV-1 PI 主要针对 成熟 PR 的催化位点，并且它们在抑制前体介导的方面明显不太有效 自动处理，表明这两种形式的 HIV-1 PR 在酶促作用上是不同的。还有，出现 PI 耐药菌株在接受含 PI 联合抗逆转录病毒疗法 (cART) 治疗的患者中的减少是一项正在进行的研究 降低治疗效果的问题，这就需要新的治疗方法。该项目旨在 找到针对当前现有药物无法识别的区域的前体的新型自动加工抑制剂 PI。为了实现这一目标，我们建立了一种基于细胞的功能测定法，忠实地概括了 用前病毒构建体观察到的自动加工表型。该测定还首次使 可以使用 AlphaLISA（放大发光接近度）通过 HTS 筛选自动加工抑制剂 均质测定（ELISA）技术。我们展示了约 26K 小分子化合物的试点屏幕 性能令人满意，Z’因子 >0.45，S/N 比 >10，命中率 <0.1%，尽管没有确认命中 被识别出来。因此，我们计划筛选一系列天然产物提取物（40K提取物，5-25 每个提取物中的化合物，总计约 60 万种化学物质），其中包含野生型和两种 PI 抗性前体 与密歇根大学生命科学研究所的 David Sherman 博士合作（目标 1）。与此同时，我们 将与博士合作。桑福德伯纳姆普雷比斯医学发现研究所的 Thomas Chung 和 Ian Pass 筛选其约 350K 小分子库（目标 2）。这些 HTS 活动有望识别出少数 已确认的化合物将接受一系列已建立的二级和三级测定（目标 3） 为了找到与目前的 HIV-1 PI 的 MOA 不同的新型自动加工抑制剂。接下来这个 新一代的治疗探针与当前的 PI 结合使用时，将实现一种新的 治疗方法：针对两种不同功能状态（前体和前体）的重要酶（HIV-1 蛋白酶） 成熟的 PR）并且同时在不同区域（非催化和催化位点）。这样的策略是 预计将大大增加 HIV-1 同时进化出可行毒株的难度（遗传障碍） 对两类抑制剂都有抵抗力以抵抗耐药性。