West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts.

中文翻译：

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黄病毒NS2B-NS3蛋白酶特异性的分析为开发选择性化学工具提供了结构基础，该工具可将登革热与寨卡病毒和西尼罗河病毒区分开。

西尼罗河病毒（WNV）和登革热病毒（DENV）是蚊子传播的致病性黄病毒。在这些病毒中发现的NS2B-NS3蛋白酶负责多蛋白加工，因此被认为是有前途的医学靶标。在Zika病毒（ZIKV）中发现了这些蛋白酶的另一个直系同源物。在这项工作中，我们应用了一种组合化学方法-混合组合底物文库（HyCoSuL），以比较在所有三种病毒中发现的NS2B-NS3蛋白酶的P4-P1位置的底物特异性谱。获得的数据表明，寨卡病毒和西尼罗河病毒NS2B-NS3蛋白酶在所有结合口袋中均显示出高度重叠的底物特异性，而登革热直向同源物对P2和P4位置的天然和非天然氨基酸的偏爱略有不同。我们使用此信息提取了登革热NS2B-NS3蛋白酶识别的特定肽序列。接下来，我们运用这一知识为登革热NS2B-NS3蛋白酶设计选择性底物和基于活性的探针。我们的工作为抑制剂的设计提供了结构框架，可用作药物开发工作的主要结构。