Highthroughput Assay for Inhibitors of HIV PR Activation

HIV PR 激活抑制剂的高通量测定

• 批准号: 7070590
• 负责人: Andrew H Kaplan
• 金额: $ 3.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070590
• 关键词: AIDS therapy; HIV infections; antiAIDS agent; antiviral agents; aspartic endopeptidases; biotechnology; drug discovery /isolation; drug screening /evaluation; enzyme linked immunosorbent assay; high throughput technology; human immunodeficiency virus 1; pharmacokinetics; protease inhibitor; technology /technique development; virus replication

DESCRIPTION (provided by applicant): Activation of the HIV-1 protease (PR) is an essential step in viral replication. Although clinically available substrate-based PR inhibitors have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. The utility of these agents is further limited by the phenomenon of cross-resistance between members of the same class. This problem appears to be especially acute for the agents that target the PR active site. Given the increasing prevalence of antiviral resistance, the somewhat limited efficacy of salvage regimens and the problems related to cross-resistance, novel approaches to inhibitor design are urgently needed. Although the available inhibitors of the HIV PR were developed using the 99 amino acid mature protease as a target, the PR is initially translated as part of the 160 kDa GagPol precursor. Processing of this precursor by the viral PR is a critical step in viral replication. As is the case for all retroviral PRs, HIV PR is only active as a homodimer and this embedded, immature PR is sufficient for precursor processing. The functional consequence of this arrangement is that the PR domains of two GagPol precursors must dimerize, become activated and begin to process the precursor in order for the viral life cycle to proceed. Mutations that interfere with the activation of the PR within GagPol have a profound effect on infectivity. We propose to develop an ELISA-based high throughput screen (HTS) for identifying compounds that inhibit the activation of the HIV PR embedded within the GagPol precursor. This system is based on our ability to express full length GagPol and monitor PR activation by measuring the release of an epitope tag. Specifically, we will: 1. Increase the sensitivity of our detection system for compounds that inhibit activity of the HIV PR embedded within GagPol. 2. Decrease the background activation of the GagPol PR.

描述（由申请人提供）：HIV-1 蛋白酶（PR）的激活是病毒复制的重要步骤。尽管临床上可用的基于底物的 PR 抑制剂对疾病进展产生了巨大影响，但在接受这些活性位点定向化合物治疗的患者中经常出现耐药变异。同一类成员之间的交叉耐药现象进一步限制了这些药物的效用。对于针对 PR 活性位点的药物来说，这个问题似乎尤其严重。鉴于抗病毒耐药性日益普遍、挽救方案的功效有限以及与交叉耐药性相关的问题，迫切需要新的抑制剂设计方法。 尽管现有的 HIV PR 抑制剂是使用 99 个氨基酸的成熟蛋白酶作为靶标开发的，但 PR 最初被翻译为 160 kDa GagPol 前体的一部分。病毒 PR 对这种前体的加工是病毒复制的关键步骤。与所有逆转录病毒 PR 的情况一样，HIV PR 仅作为同二聚体具有活性，并且这种嵌入的、未成熟的 PR 足以进行前体加工。这种排列的功能结果是两个 GagPol 前体的 PR 结构域必须二聚化、被激活并开始处理前体，以便病毒生命周期继续进行。干扰 GagPol 内 PR 激活的突变对感染性具有深远影响。我们建议开发一种基于 ELISA 的高通量筛选 (HTS)，用于识别抑制 GagPol 前体中嵌入的 HIV PR 激活的化合物。该系统基于我们表达全长 GagPol 并通过测量表位标签的释放来监测 PR 激活的能力。具体来说，我们将： 1. 提高我们的检测系统对抑制 GagPol 中嵌入的 HIV PR 活性的化合物的灵敏度。 2. 减少 GagPol PR 的背景激活。