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，但在接受这些活性部位定向化合物治疗的患者中经常出现抗性变异。这些药物的效用进一步受到同一阶级成员之间交叉抗性现象的限制。对于针对PR活动位点的代理人来说，这个问题似乎特别急切。鉴于抗病毒抗性的患病率的增加，迫切需要迫切需要打捞方案的疗效和与跨耐药性有关的问题，因此迫切需要抑制剂设计的新方法。 尽管使用99个氨基酸成熟蛋白酶作为靶标开发了HIV PR的可用抑制剂，但最初将PR作为160 kDa Gagpol前体的一部分翻译而成。通过病毒PR处理该前体是病毒复制的关键步骤。与所有逆转录病毒PR一样，HIV PR仅作为同型二聚体活性，而这种嵌入的，未成熟的PR足以进行前体处理。这种安排的功能后果是，两个Gagpol前体的PR结构域必须降低，激活并开始处理前体，以使病毒生命周期进行。干扰GAGPOL中PR激活的突变对感染性具有深远的影响。我们建议开发基于ELISA的高吞吐量屏幕（HTS），以识别抑制Gagpol前体中嵌入的HIV PR激活的化合物。该系统基于我们通过测量表位标签的释放来表达全长Gagpol和监视PR激活的能力。具体来说，我们将： 1。提高我们检测系统对抑制Gagpol中HIV PR活性的化合物的敏感性。 2。减少Gagpol PR的背景激活。