Assay Optimization for Identification of Novel HIV-1 Protease Autoprocessing Inhi

项目概述：本研究旨在优化一种基于细胞的功能分析方法，用于高通量筛选选择性抑制HIV-1蛋白酶自动加工的小分子，这是一种重要的病毒特异性过程，尚未用于抗hiv药物开发。在被感染的细胞中，HIV蛋白酶最初作为Gag-Pol多蛋白前体的一部分被合成。在病毒粒子生产的后期，前体催化裂解反应，导致自由的、完全活跃的、成熟的蛋白酶的解放。目前FDA批准的蛋白酶抑制剂（pi）主要针对成熟蛋白酶的催化位点。然而，这些pi在抑制前体自动加工方面的效果明显较差，这表明这两种形式的HIV-1蛋白酶在酶上并不相同。我们最近开发了一种测定前体自动处理的定量方法。该方法也有潜力用于AlphaLISA（放大发光接近均质法ELISA）的高通量筛选（HTS）新型自动处理抑制剂。试验筛管的z′系数在0.5 ~ 0.7之间，信噪比大于100。在此基础上，我们建议建立/优化可用于新型自动处理抑制剂HTS的主要分析条件（Aim 1）；并通过小规模筛选评估初级试验（目标2）。这些研究结果将为通过大规模筛选鉴定新的自动加工抑制剂奠定基础。对已鉴定化合物的进一步表征将有助于开发一种急需的新型治疗药物，这种药物可以在不同于当前pi靶向的区域/阶段靶向HIV-1蛋白酶。将这种新一代的自动加工抑制剂与目前的治疗方案结合起来，可能会显著改善艾滋病毒感染者的治疗效果。这些新药的生化和结构检测也有助于揭示前体自动加工的机制。

DESCRIPTION (provided by applicant): Assay Optimization for Identification of Novel HIV-1 Protease Autoprocessing Inhibitors Project Summary This study aims to optimize a cell-based functional assay for high-throughput screens of small molecules that selectively suppress HIV-1 protease autoprocessing, an essential viral-specific process that has not been 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 catalyzes the cleavage reactions that lead to the liberation of the free, fully active, mature protease. The currently available FDA- approved protease inhibitors (PIs) primarily target the mature protease at its catalytic site. These PIs, however, are significantly less effective at suppressing precursor autoprocessing, suggesting that these two forms of HIV-1 protease are not enzymatically identical. We have recently developed an assay for quantification of precursor autoprocessing. This assay also has the potential to be used for high-throughput screen (HTS) of novel autoprocessing inhibitors with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Our pilot screen demonstrated a z' factor ranging from 0.5 to 0.7 and S/N ratios greater than 100. Built upon this platform, we here propose to establish/optimize the primary assay conditions that can be used for HTS of novel autoprocessing inhibitors (Aim 1); and to evaluate the primary assay via a small-scale screen (Aim 2). Results of these developments will lay the foundation for identification of novel autoprocessing inhibitors through large scale screens. Further characterization of the identified compounds will aid in the development of a much-needed new class of therapeutic drugs that target the HIV-1 protease at regions/stages different from those targeted by the current PIs. A cocktail combining this next generation of autoprocessing inhibitors with the current regimen may significantly improve the treatment outcomes of those living with HIV. Biochemical and structural examination of these new drugs may also shed light on the mechanism of precursor autoprocessing.