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

描述（由申请人提供）：用于鉴定新型HIV-1蛋白酶自动加工抑制剂的试验优化项目总结本研究旨在优化基于细胞的功能性试验，用于选择性抑制HIV-1蛋白酶自动加工的小分子的高通量筛选，这是一种重要的病毒特异性过程，尚未用于抗HIV药物开发。在受感染的细胞中，HIV蛋白酶最初作为Gag-Pol多蛋白前体的一部分合成。在病毒体产生的后期阶段，前体催化裂解反应，导致游离的、完全活性的成熟蛋白酶的释放。目前可获得的FDA批准的蛋白酶抑制剂（PI）主要靶向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有效性显著降低，这表明这两种形式的HIV-1蛋白酶在酶促作用上并不相同。我们最近开发了一种用于定量前体自动加工的测定法。该测定也有可能用于AlphaLISA（扩增发光邻近均相测定ELISA）的新型自加工抑制剂的高通量筛选（HTS）。我们的试验筛选显示z'因子范围为0.5至0.7，S/N比大于100。基于这个平台，我们在这里建议建立/优化可用于新型自加工抑制剂HTS的初步测定条件（目的1）;并通过小规模筛选（目的2）评估初步测定。这些研究结果将为通过大规模筛选鉴定新型自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急需的新型治疗药物，这些药物靶向HIV-1蛋白酶的区域/阶段与当前PI靶向的区域/阶段不同。将这种下一代自动加工抑制剂与当前方案相结合的鸡尾酒可能会显着改善艾滋病毒感染者的治疗结果。这些新药的生化和结构检测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.