Novel Assay for HIV-1 Integrase Inhibitors

描述（由申请人提供）：HIV-1表型对当前可用药物的不断出现，表明需要开发具有替代作用机制的新疗法。在目前的应用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相互作用的IN亚基的高度动态性质。有趣的是，晶状体上皮衍生生长因子（LEDGF）的整合酶结合域（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IN亚基-亚基相互作用。这种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学上必要的协调整合步骤。这些观察结果揭示了抑制HIV-1 IN功能的潜在新机制，并为以下假设提供了令人信服的理论依据：高度灵活的多聚形式HIV-1 IN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。为了解决这个前提，我们提出以下两个主要目标。目的1将识别和表征不同多聚形式的IN中可能被小分子抑制剂靶向的新“热点”。创新的质谱蛋白质足迹和分子建模方法将在这些研究中使用。目标2将开发并验证一种用于新型HIV-1整合酶抑制剂高通量筛选（HTS）的新型荧光测定法。该检测将监测亚基-亚基之间的相互作用亲和力标签含有和无标签荧光标记的蛋白质。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。两种蛋白质亚基之间的生产性交换将产生强烈的荧光，而这种反应的有效抑制剂将显著减弱荧光信号。实验将通过小化学文库的初步筛选进行优化。这些研究将确定重要的统计参数和有效的方案，为未来大型化学文库HTS的新型荧光分析应用。

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.