Structure and function of dengue proteases

在登革病毒(DEN)感染期间，病毒-病毒和病毒-宿主蛋白的相互作用对 病毒后代的产生和宿主免疫反应的调节。其中任何一项的破坏 相互作用可能对病毒的生命周期有害。因此，化合物的鉴定能够 破坏这些相互作用对于开发针对DEN的有效治疗方法至关重要 感染。我们建议为小分子的设计、优化和测试奠定一个框架 DEN的抑制剂(图1)。我们将利用一个正在进行的发展项目(始于2008年3月)，旨在 在鉴定病毒蛋白酶NS3的抑制化合物时，通过获得与NS3一起的额外结构 与候选抑制剂的关联。然后对候选化合物进行先导优化， PSWRCE的合作者正在进行疗效验证。我们还将进行结构调整 已知相互作用伙伴络合物中DEN蛋白的测定。然后，结构信息将被 用于设计抑制性化合物或筛选抑制性化合物的方法。最后，我们会 确定将作为共结晶和抑制物靶标的其他DEN蛋白质-蛋白质相互作用 设计。 具体目标1：继续和扩大正在进行的基于结构的药物设计工作，使用来自 所有四种血清型。我们将使用分子筛进行高通量筛查分析 图书馆筛选中心网络和商业上可用的基于片段的筛选，以识别新的 先导化合物。然后我们将与这些化合物获得共晶结构，以指导合理的抑制剂 设计。 具体目标2：对与DEN有关的已确定的蛋白质-蛋白质相互作用进行结构研究 感染。 具体目标3：确定用于结构研究的其他病原体-宿主蛋白质-蛋白质相互作用 亲和纯化和质谱分析以确定蛋白质中存在的相互作用伙伴 从哺乳动物细胞中分离出来的复合体。

During a Dengue virus (DEN) infection, viral-viral and viral-host protein interactions are crucial for the generation of viral progeny and modulation of the host immune response. Disruption of any of these interactions is potentially detrimental to the viral life cycle. Hence, the identification of compounds capable of disrupting these interactions is critically important for the development of effective therapies against DEN infections. We propose to lay a framework for the design, optimization, and testing of small molecule inhibitors for DEN (Fig. 1). We will leverage an ongoing developmental project (started in March 2008) aimed at identifying inhibitory compounds for the viral protease, NS3, by obtaining additional structures with NS3 in association with candidate inhibitors. Lead optimization of candidate compounds will then be performed with efficacy validation being carried out by collaborators in the PSWRCE. We will also conduct structure determination of DEN proteins in complex with known interacting partners. Structural information will then be used to design inhibitory compounds or assays designed to screen for inhibitory compounds. Finally, we will identify additional DEN protein-protein interactions that will serve as targets for co-crystallization and inhibitor design. Specific Aim 1: Continue and extend ongoing structure-based drug design efforts with DEN proteases from all four serotypes. We will perform high-throughput screening assays with screens from the Molecular Libraries Screening Centers Network and commercially available fragment-based screens to identify new lead compounds. We will then obtain co-crystal structures with these compounds to guide rational inhibitor design. Specific Aim 2: Conduct structural studies on identified protein-protein interaction involved in DEN infections. Specific Aim 3: Identify additional pathogen-host protein-protein interactions for structural studies by utilizing affinity purification followed by mass spectrometry analysis to identify interacting partners present in protein complexes isolated from mammalian cells.