Novel Assay for HIV-1 Integrase Inhibitors

描述(由申请人提供)：对现有药物产生抗药性的HIV-1表型的不断出现要求开发具有替代作用机制的新疗法。本申请中提出的一种这样的机制是利用关键的HIV-1酶整合酶(IN)的多聚体结构。我们最近的研究揭示了亚基相互作用的高度动态性质。有趣的是，晶状体上皮源性生长因子(LEDGF)的整合酶结合结构域(IBD)是HIV-1整合的关键细胞辅助因子，通过将游离病毒蛋白“锁定”到四聚体状态，深刻地改变了亚基-亚单位之间的相互作用。这个IN四聚体在3‘-加工反应中是活跃的，但不能催化第二个生物必需的协同整合步骤。这些观察揭示了一种潜在的抑制HIV-1 IN功能的新机制，并为以下假设提供了令人信服的理论基础：高度灵活的HIV-1 IN多聚体具有独特的结构口袋，可以被小分子选择性地靶向以抑制协同整合反应。为了解决这一前提，我们提出了以下两个主要目标。目标1将识别和表征不同多聚体形式的IN中可能成为小分子抑制剂靶标的新的“热点”。在这些研究中将使用创新的质谱学蛋白质足迹和分子建模方法。AIM 2将开发和验证一种新的荧光分析方法，用于高通量筛选新型HIV-1整合酶抑制剂。该分析将监测含有亲和标签的和无标签的荧光标记蛋白之间的亚基-亚基相互作用。相互作用的蛋白质将被亲和树脂拉下来，并通过荧光测量进行评估。这两个蛋白质亚基之间的生产性交换将导致强烈的荧光，而这种反应的有效抑制剂将显著减弱荧光信号。将通过对小化学文库的初步筛选来优化检测方法。这些研究将确定重要的统计参数和一个有效的方案，为新的荧光分析方法在大型化学库的高温超导中的应用奠定基础。 公共卫生相关性：出现对当前抗逆转录病毒疗法产生抗药性的HIV-1毒株是一个严重的临床问题。因此，迫切需要识别和验证用于药物发现的新的病毒靶点。本提案中研究的一个这样的目标是关键的HIV-1酶整合酶的多聚体结构。

DESCRIPTION (provided by applicant): The continuous emergence of HIV-1 phenotypes resistant to the currently available drugs dictates a need to develop new therapies with alternative mechanisms of action. One such mechanism proposed in the present application is to exploit multimeric structures of a key HIV-1 enzyme integrase (IN). Our recent studies revealed a highly dynamic nature of interacting IN subunits. Interestingly, the integrase binding domain (IBD) of lens epithelium derived growth factor (LEDGF), a key cellular cofactor for HIV-1 integration, profoundly alters IN subunit-subunit interactions by "locking" the free viral protein into a tetrameric state. This IN tetramer is active in the 3'-processing reaction but fails to catalyze the second biologically essential concerted integration step. These observations uncovered a potential novel mechanism of inhibiting the HIV-1 IN function and provided a compelling rationale for the following hypothesis: highly flexible multimeric forms of HIV-1 IN possess unique structural pockets that can be selectively targeted by small molecules to inhibit the concerted integration reaction. To address this premise we propose the following two principal aims. Aim 1 will identify and characterize novel "hotspots" within different multimeric forms of IN that can potentially be targeted by small molecule inhibitors. Innovative mass spectrometric protein footprinting and molecular modeling approaches will be used in these studies. Aim 2 will develop and validate a novel fluorescence assay for high-throughput screening (HTS) of new types of HIV-1 integrase inhibitors. The assay will monitor subunit-subunit interactions between affinity tag containing and tag-free fluorescently labeled proteins. The interacting proteins will be pulled down by the affinity resin and evaluated by fluorescence measurements. The productive exchange between the two protein subunits will result in strong fluorescence, while potent inhibitors of this reaction will significantly diminish the fluorescence signal. The assay will be optimized through initial screening of small chemical libraries. These studies will define important statistical parameters and an effective protocol for future application of the novel fluorescence assay for HTS of large chemical libraries. PUBLIC HEALTH RELEVANCE: Emergence of HIV-1 strains resistant to the current antiretroviral therapies is a serious clinical problem. Therefore, there is an urgent need to identify and validate new viral targets for drug discovery. One such target investigated in the present proposal is a multimeric structure of a key HIV-1 enzyme integrase.