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

描述(由申请人提供)：鉴定新型HIV-1蛋白酶自动处理抑制剂的测试优化项目摘要本研究旨在优化一种基于细胞的功能分析，用于高通量筛选选择性抑制HIV-1蛋白酶自动处理的小分子，这是一种基本的病毒特异性过程，尚未被用于抗HIV药物的开发。在感染的细胞中，HIV蛋白酶最初是作为Gag-Pol多蛋白前体的一部分合成的。在病毒粒子产生的后期，前体催化裂解反应，导致自由的、充分活跃的、成熟的蛋白酶的释放。目前已有的FDA批准的蛋白水解酶抑制剂(PI)主要针对成熟的蛋白酶的催化部位。然而，这些PI在抑制前体自动处理方面的效果明显较差，这表明这两种形式的HIV-1蛋白酶在酶学上并不相同。我们最近开发了一种对前体自动加工进行量化的分析方法。该方法也有可能用于高通量筛选(HTS)新的自动处理抑制剂与AlphaLISA(放大发光均相分析)。我们的试点屏幕显示的z‘因子范围在0.5到0.7之间，S/N比大于100。在此平台的基础上，我们建议建立/优化可用于新型自动处理抑制剂HTS的初步检测条件(目标1)；并通过小规模筛选对初步检测进行评估(目标2)。这些进展的结果将为通过大规模筛选鉴定新型自动处理抑制剂奠定基础。对已确定化合物的进一步表征将有助于开发急需的新一类治疗药物，这些药物以HIV-1蛋白酶为靶点，其区域/阶段不同于当前PIs的靶点。将这种下一代自动处理抑制剂与当前方案相结合的鸡尾酒可能会显著改善艾滋病毒携带者的治疗结果。对这些新药的生化和结构检查也可能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.