Assay Development for Identification of HIV-1 Protease Autoprocessing Specific Inhibitors

HIV-1蛋白酶自动加工特异性抑制剂检测方法的建立 项目摘要 本提案是对PAR-13-364：开发用于以下用途的高通量筛选试验的回应 探针和治疗前发现。本研究旨在建立一种多重初步检测方法， 开发和评估后续检测方法，以便能够识别选择性靶向HIV的小分子， 1蛋白酶前体自动加工，这是一种以前尚未开发的重要病毒特异性过程 用于抗艾滋病药物的研发。在受感染的细胞中，HIV蛋白酶最初是作为Gag-Pol的一部分合成的。 多蛋白前体在病毒体产生的后期，前体自催化裂解 目前可获得的FDA批准的蛋白酶抑制剂， (PIs)这些PI主要针对成熟PR的催化位点。这些PI在抑制PR方面的效果明显较差， 前体自动加工，这表明这两种形式的HIV-1蛋白酶是不同的酶。这 拟议的研究将开发和建立一个平台，使HTS运动寻找新的 作用模式与当前PI不同的自动处理抑制剂。下一代治疗药物 探针，当与目前的PI结合使用时，将实现一种新的治疗方法：靶向 在两种不同的功能状态（获得和成熟PR）和不同区域的重要酶（HIV-1蛋白酶 （非催化位点和催化位点）。这样的战略预计将大幅增加难度 （遗传屏障）使HIV进化出同时对两类抑制剂具有抗性的可行菌株。 我们最近建立了一种基于细胞的检测方法，用于研究HIV-1蛋白酶的自动加工机制。 该测定首次使得通过AlphaLISA（扩增的ELISA）筛选自动加工抑制剂成为可能。 发光邻近均相测定ELISA）技术。我们的试验筛选了23,000个小分子 化合物显示Z'因子>0.5和S/N比>15，并鉴定了35个高活性命中， 抑制活性接近阳性对照（图7）。基于这一平台，我们在此提议， 进一步将主要测定开发为多重形式，以实现自动处理的标准化定量 转染细胞数（Aim 1a）的效率。该更新的多重初步试验将用于 用一种新的融合前体来筛选20，000种生命多样性化合物的集合， 用前病毒模型观察到的表型（目的1b）。从Aim 1b和我们之前的研究中发现的高度活跃的命中 将使用连续稀释的新鲜储备化合物（目标1c）验证中试筛选，并确认高优先级 命中将受到一系列二级和三级检测，以检查其可能的 作用机制（目标2a-d）。特别是，我们希望鉴定出抑制前体的化合物， 通过与成熟PR的已知催化位点以外的区域相互作用进行自动加工（即，变构 抑制剂）。新的自动加工抑制剂将进一步评估其对复制的抑制作用 HIV-1毒株（Aim 2f）。这些拟议的实验的成功执行将建立一个框架， 通过大规模筛选鉴定新的自动加工特异性抑制剂。的进一步表征 所鉴定的化合物将有助于开发一类新的治疗药物， 当前PI对这些新药的生化和结构检查也可能揭示其机制 自动处理的先驱。

Assay Development for Identification of HIV-1 Protease Autoprocessing Specific Inhibitors Project Summary This proposal is in response to PAR-13-364: Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery. The proposed study aims to establish a multiplex primary assay and develop and evaluate follow-up assays that will enable identification of small molecules selectively targeting HIV- 1 protease precursor autoprocessing, an essential viral-specific process that has not been previously exploited for anti-HIV drug development. In the infected cell, HIV protease is initially synthesized as part of the Gag-Pol polyprotein precursor. During the late stage of virion production, the precursor self-catalyzes the cleavage reactions that lead to liberation of the free mature PR. The currently available FDA-approved protease inhibitors (PIs) primarily target the catalytic site of the mature PR. These PIs are significantly less effective at suppressing precursor autoprocessing, suggesting that these two forms of HIV-1 protease are enzymatically different. This proposed study will develop and establish a platform to enable HTS campaigns to search for novel autoprocessing inhibitors with action modes different from the current PIs. 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 (procure and mature PR) and at different regions (non-catalytic site and catalytic site) at the same time. Such a strategy is expected to drastically increase difficulty (genetic barrier) for HIV to evolve viable strains simultaneously resistant to inhibitors from both classes. We have recently established a cell-based assay for the study of HIV-1 protease autoprocessing mechanism. This assay has for the first time made it possible to screen for autoprocessing inhibitors by AlphaLISA (amplified luminescent proximity homogeneous assay ELISA) technology. Our pilot screens of 23,000 small molecule compounds displayed a Z' factor >0.5 and an S/N ratio >15, and identified 35 highly active hits displaying inhibition activities approaching the positive controls (Figure 7). Built upon this platform, we here propose to further develop the primary assay into a multiplex format to enable normalized quantification of autoprocessing efficiency to the numbers of transfected cells (Aim1a). This updated multiplex primary assay will be used to screen a collection of 20,000 life diversity compounds with a new fusion precursor that mimics an autoprocessing phenotype observed with a proviral model (Aim 1b). The highly active hits found from Aim 1b and our previous pilot screen will be verified with fresh stock compounds in serial dilutions (Aim 1c) and the confirmed high priority hits will be subjected to a battery of secondary and tertiary assays to be established to examine their possible action mechanisms (Aims 2a-d). In particular, we hope to identify compounds that inhibit precursor autoprocessing by interacting with a region(s) beyond the known catalytic site of the mature PR (i.e., allosteric inhibitors). The novel autoprocessing inhibitors will be further evaluated for their inhibitory effects on replication of HIV-1 strains (Aim 2f). Successful execution of these proposed experiments will establish a framework for identification of novel autoprocessing specific inhibitors through large scale screens. Further characterization of the identified compounds will aid in the development of a new class of therapeutic drugs to complement the current PIs. Biochemical and structural examination of these new drugs may also shed light on the mechanism of precursor autoprocessing.