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

HTS靶向HIV-1蛋白酶自动加工，用于一流的药物发现 项目摘要 本提案是对PAR-17-438：用于发现化学探针的检测开发和筛选的响应 或治疗剂（R 01）。本项目的目标是进行高通量筛选和后续 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 在一些实施方案中，本发明涉及与目前可用的蛋白酶抑制剂（PI）不同的MOA。在受感染的细胞中，HIV-1 PR最初是 作为Gag-Pol多聚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 在病毒体产生的后期阶段，前体自催化裂解反应，导致病毒体的释放。 以时空调节的方式释放成熟PR。FDA批准的HIV-1 PI主要针对 在成熟PR的催化位点上，它们在抑制受体介导的 自动加工，这表明这两种形式的HIV-1 PR是不同的酶。此外， 在接受含PI的联合抗逆转录病毒治疗（cART）的患者中，PI耐药株的研究正在进行中。 降低治疗效果的问题，这保证了对新疗法的需要。本项目谋求 发现新的自动加工抑制剂，靶向目前可用的未识别的区域的前体 私家侦探为了实现这一目标，我们建立了一个基于细胞的功能测定，忠实地概括了 用前病毒构建体观察到的自动加工表型。这项试验也首次使 可以使用AlphaLISA（放大的发光邻近度）通过HTS筛选自动加工抑制剂 均相测定ELISA）技术。我们的~ 26 K小分子化合物的中试筛选显示 令人满意的性能，Z'因子>0.45，S/N比>10，命中率<0.1%，但未确认命中 被确认了因此，我们计划筛选一系列天然产物提取物（40 K提取物，5-25 化合物，总计约60万种化学物质），其中野生型和两种PI抗性前体在 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医学发现研究所的托马斯钟博士和伊恩帕斯博士合作， 筛选其~ 350 K小分子文库（Aim 2）。这些HTS活动将有望确定少数 已确认的化合物将在以下研究中进行一系列已建立的二级和三级试验（目标3）： 为了找到新的自加工抑制剂，是从目前的HIV-1 PI在其MOA不同。下一 新一代治疗探针与当前PI结合使用时，将实现新的 治疗方法：以两种不同功能状态（前体和 成熟的PR）和不同的区域（非催化和催化位点）。这样的策略 预计将大大增加HIV-1同时进化出可行菌株的难度（遗传障碍） 对来自两类的抑制剂具有抗性以抵抗抗性。

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.