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

针对HIV-1蛋白酶自动处理的HTS用于一流的药物发现 项目摘要 这项建议是对PAR-17-438的回应：化验的发展和筛选化学探针的发现 或治疗剂(R01)。该项目的目标是进行高通量筛选和后续 用作用模式鉴定抑制HIV-1蛋白水解酶(PR)自动加工的分子 (MOAS)不同于目前可用的蛋白酶抑制剂(PI)。在被感染的细胞中，HIV-1 PR最初是 作为GAG-POL多蛋白前体的一部分合成，其蛋白分解活性被严格抑制。在.期间 在病毒粒子产生的后期，前体自我催化切割反应，导致释放 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的催化部位，它们抑制前体介导的效果明显较差 自动处理，这表明这两种形式的HIV-1PR在酶学上是不同的。此外，新出现的 在接受含PI的联合抗逆转录病毒疗法(CART)治疗的患者中，PI耐药株的评估正在进行中 这一问题降低了治疗效果，需要新的治疗方法。这一项目旨在 寻找针对前体的新的自动处理抑制剂，该区域不被当前可用的 PIS.为了实现这一目标，我们建立了一种基于细胞的功能分析方法，它忠实地概括了 观察到的前病毒结构的自动加工表型。这项测试也第一次成功地 可以通过使用AlphaLISA(放大发光接近度)的HTS来筛选自动处理抑制物 均质检测)技术。我们展示了~26K个小分子化合物的中试屏幕 令人满意的表现，Z‘因素&0.45；，S/N比率&10，命中率&0.1%，尽管没有确认的命中 已被确认。因此，我们计划筛选一系列天然产品提取物(40K提取物，5-25 每种提取物含有化合物，总计约60万种化学物质)，其中包括一种野生型和两种抗PI前体 与密歇根大学生命科学研究所的大卫·谢尔曼博士合作(目标1)。同时，我们 将与Sanford Burnham Prebys医学发现研究所的Thomas Chung博士和Ian Pass博士合作 筛选其~350K小分子文库(目标2)。这些HTS活动有望确定少数几个 已确认的化合物将接受一系列已建立的二次和三次分析(目标3) 以寻找不同于目前的HIV-1在其MOA中的PI的新的自动处理抑制剂。接下来的这个 当与当前的PI结合使用时，新一代治疗性探针将实现新的 治疗方法：针对两种不同功能状态的重要酶(HIV-1蛋白酶)(前体和 成熟的PR)，同时在不同的区域(非催化和催化部位)。这样的战略是 预计将大幅增加HIV-1同时进化存活毒株的难度(遗传障碍) 抵抗来自两个类别的抑制剂以抵抗耐药性。

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.