Chemical Tools for the Study of Dengue Virus Entry

描述(申请人提供)：登革热病毒感染造成的巨大负担，加上缺乏有效的疫苗或药物，使开发新的治疗方法成为高度优先事项。我们之前已经确定了二取代的嘧啶类化合物，它通过与病毒粒子表面融合前的E二聚体结合来抑制登革热病毒的进入，可能是在Harison和他的同事之前发现的β-八糖苷结合口袋中。在这里，我们建议开发检测方法，以确定该位点的其他配体可以抑制登革热病毒的进入。随后的高通量筛选(HTS)工作和下游活动需要询问可以结合在这个口袋中的化学多样性，产生更多的先导化合物，并阐明有效的抗登革热活性的结构和机制相关性。在目标1中，我们将使用这些化合物开发一种高通量荧光偏振分析方法，以确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，我们建议建立一种在体外监测病毒颗粒整体融合的方法。在目标3中，我们将通过进行中试筛选来验证我们的分析流水线。我们将使用AIMS 1和AIMS 2中开发的分析方法以及我们实验室以前开发的其他分析方法来表征具有抗登革热病毒活性的化合物的作用机制。在这项建议的结论，我们预计有强大的分析，准备高通量筛查。这些下游HTS努力的目标是开发分子探针，以研究登革热进入的结构和生化机制，验证β-辛糖苷口袋作为抑制登革热进入的分子靶点，并确定控制β-辛糖苷口袋配体的抗登革热活性的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.