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测定优化 项目摘要 本研究旨在优化基于细胞的功能测定，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。在病毒颗粒产生的后期，前体催化裂解反应，导致释放出游离的、完全活性的、成熟的蛋白酶。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，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前体自动加工定量分析方法。该测定还有可能用于使用 AlphaLISA（放大发光邻近均相测定 ELISA）对新型自动加工抑制剂进行高通量筛选 (HTS)。我们的试点筛选显示了 z' 因子范围为 0.5 至 0.7，信噪比大于 100。基于此平台，我们在此建议建立/优化可用于新型自动加工抑制剂的 HTS 的主要测定条件（目标 1）；并通过小规模筛选评估初步测定（目标 2）。这些进展的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一类急需的新型治疗药物，该药物以不同于当前 PI 靶向的区域/阶段的 HIV-1 蛋白酶为目标。将下一代自动加工抑制剂与当前治疗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.