Chemical Tools for the Study of Dengue Virus Entry

描述（由申请人提供）：登革热病毒感染导致的严重负担加上缺乏有效的疫苗或药物，使得开发新的治疗方法成为高度优先事项。我们先前已经鉴定了二取代的嘧啶，其通过与病毒体表面上的融合前E二聚体结合来抑制登革病毒进入，可能在先前由Harrison及其同事鉴定的β-葡糖苷结合口袋中。在这里，我们建议开发检测，以确定其他配体的这个网站，可以抑制登革病毒进入。随后的高通量筛选（HTS）的努力和下游活动需要询问的化学多样性，可以结合在这个口袋里，产生额外的先导化合物，并阐明有效的抗登革热活性的结构和机制的相关性。在目标1中，我们将使用这些化合物来开发高通量荧光偏振分析，以鉴定抑制登革病毒感染的登革病毒包膜蛋白的配体。在目标2中，我们提出开发一种检测方法来监测病毒颗粒在体外的大量融合。在目标3中，我们将通过进行试点筛选来验证我们的检测管道。我们将使用目标1和2中开发的测定以及我们实验室先前开发的其他测定来表征具有抗登革热病毒活性的化合物的作用机制。在这个建议的结论，我们预计有强大的检测，准备高通量筛选。这些下游HTS努力的目标是开发分子探针以研究登革进入的结构和生化机制，验证β-葡糖苷口袋作为抑制登革进入的分子靶标，并定义控制β-葡糖苷口袋配体的抗登革活性的SAR。从长远来看，这项工作也将有助于我们未来的目标，即开发DV进入的小分子抑制剂，作为抗登革热药物发现工作的临床前候选药物。

DESCRIPTION (provided by applicant): The significant burden that results from dengue virus infection combined with the absence of effective vaccines or drugs makes the development of novel therapeutics a high priority. We have previously identified disubstituted pyrimidines that inhibit dengue virus entry by binding to the pre-fusion E dimer on the virion surface, perhaps in the beta-octoglucoside binding pocket previously identified by Harrison and colleagues. Here, we propose to develop assays to identify other ligands of this site that can inhibit dengue virus entry. The subsequent high-throughput screening (HTS) effort and downstream activities are needed to interrogate the chemical diversity that can bind in this pocket, to generate additional lead compounds, and to elucidate the structural and mechanistic correlates of potent anti-dengue activity. In Aim 1, we will use these compounds to develop a high-throughput fluorescence polarization assay to identify ligands of the dengue virus envelope protein that inhibit dengue virus infection. In Aim 2, we propose to develop an assay to monitor bulk fusion of viral particles in vitro. In Aim 3, we will validate our assay pipeline by performing a pilot screen. We will use assays developed in Aims 1 and 2 as well as other assays previously developed in our laboratory to characterize the mechanisms of action of compounds with activity against dengue virus. At the conclusion of this proposal, we anticipate having robust assays that are ready for high-throughput screening. The goal of these downstream HTS efforts is the development of molecular probes to study the structural and biochemical mechanisms of dengue entry, to validate the beta-octoglucoside pocket as a molecular target for inhibition of dengue entry, and to define the SAR governing the anti-dengue activity of ligands of the beta-octoglucoside pocket. In the longer term, this work will also contribute to our future goals of developing small molecule inhibitors of DV entry as preclinical candidates for anti-dengue drug discovery efforts.