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

新型 HIV-1 蛋白酶自动加工酶鉴定的测定优化

• 批准号: 8789526
• 负责人: CHAOPING CHEN
• 金额: $ 7.44万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-16 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8789526
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Anti-HIV Agents; Antiviral Agents; Binding; Biochemical; Biological Assay; Catalytic Domain; Cells; Detection; Development; Drug Targeting; Drug resistance; Enzyme-Linked Immunosorbent Assay; Epidemic; FDA approved; Foundations; Future; Gagging; Generations; Goals; HIV; HIV Protease; HIV drug resistance; HIV-1; Infection; Lead; Libraries; Life; Light; Mammalian Cell; Mediating; Medicine; Molecular; Molecular Bank; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Process; Production; Protease Domain; Protease Inhibitor; Reaction; Regimen; Research; Sagittaria; Staging; Structure; Testing; Therapeutic; Time; Treatment Efficacy; Treatment outcome; Viral; Virion; Virus; analog; base; drug development; high throughput screening; improved; inhibitor/antagonist; innovation; next generation; novel; pol Gene Products; public health relevance; small molecule; stable cell line; therapeutic target; tool

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.

描述（由申请人提供）：用于识别新型HIV-1蛋白酶自动处理抑制剂项目摘要的测定优化本研究旨在优化针对小分子的高通量筛选的基于细胞的功能测定，这些分子选择性地抑制HIV-1蛋白酶自动化过程，这是一种基本的病毒特异性过程，这是一种未利用抗毒药的基本病毒特异性过程。在感染的细胞中，HIV蛋白酶最初合成为GAG-POL多蛋白前体的一部分。在病毒素产生的后期，前体催化了裂解反应，从而导致自由，充分活跃，成熟的蛋白酶解放。当前可用的FDA批准蛋白酶抑制剂（PI）主要针对成熟的蛋白酶在其催化部位。然而，这些PI在抑制前体自动加入方面的有效性明显较小，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用于定量前体自动加入的测定法。该测定法也有可能用于与α的新型自动化抑制剂的高通量屏幕（HTS）（放大发光近端均匀均匀测定ELISA）。我们的试点屏幕显示Z'因子范围为0.5至0.7至0.7，S/N比大于100。构建在该平台上，我们在这里建议建立/优化可用于新型自动化抑制剂HTS的主要测定条件（AIM 1）；并通过小规模屏幕评估主要测定法（AIM 2）。这些发展的结果将奠定基础，以通过大规模筛选来识别新型的自动化抑制剂。确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，该药物针对的是在区域/阶段的HIV-1蛋白酶，与当前PI的靶向不同。将下一代自动加入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患者的治疗结果。这些新药的生化和结构检查也可能阐明前体自动加入的机制。