Cleaving through complexity: Pan-serotype fluorimetric assays for the dengue virus proteases
Dengue virus (DENV) is a health threat with global impact. The viral NS2B/NS3 protease is responsible for viral polyprotein processing at a number of cleavage sites. This cleavage is indispensable for establishing productive infection, highlighting the pivotal role of the DENV protease. As such this...
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| Main Authors: | , , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
August 2026
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| In: |
SLAS discovery
Year: 2026, Volume: 41, Pages: 1-13 |
| ISSN: | 2472-5560 |
| DOI: | 10.1016/j.slasd.2026.100310 |
| Online Access: | Verlag, kostenfrei, Volltext: https://doi.org/10.1016/j.slasd.2026.100310 Verlag, kostenfrei, Volltext: https://www.sciencedirect.com/science/article/pii/S247255522600016X 
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| Author Notes: | Miguel M. Macedo, Johannes Lang, Katharina Eckstein, Selina L. Muffler, Leah M. Glanzmann, Christian Klein, Mila-M. Leuthold |
| Summary: | Dengue virus (DENV) is a health threat with global impact. The viral NS2B/NS3 protease is responsible for viral polyprotein processing at a number of cleavage sites. This cleavage is indispensable for establishing productive infection, highlighting the pivotal role of the DENV protease. As such this enzyme is a promising target for the development of antivirals. The absence of a standardized enzymatic assay procedure, using diverse assay conditions and protease constructs, poses a challenge in evaluation of inhibition data. Here, we describe the analysis and comparison of assay parameters for proteases of all four circulating DENV1 to DENV4 serotypes, eventually enabling high-throughput screening (HTS). DENV1-4 proteases were used in the detailed exploration of biochemical assay conditions and a number of FRET substrates as well as AMC-coupled fluorogenic substrates were characterized. The DENV1 protease showed the lowest levels of substrate cleavage, with kcat values several folds below the others (0.03 s-1 vs. 0.4 s-1). This prompted us to design novel substrates based on the 2A/2B and 2B/3 cleavage sites. Enzyme kinetics and inhibition of the DENV1-4 proteases were characterized under two widely applicable assay conditions. The feasibility to use these conditions for HTS was verified in 384-well format. Based on the results, we present two robust assay protocols, suitable to examine activity and inhibition of proteases of all four DENV serotypes in parallel, allowing for HTS. A well-characterized assay procedure can enhance coherence in DENV protease studies, contributing to progress in antiviral research. |
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| Item Description: | Online verfügbar 28 April 2026, Version des Artikels 14 May 2026 Gesehen am 30.07.2026 |
| Physical Description: | Online Resource |
| ISSN: | 2472-5560 |
| DOI: | 10.1016/j.slasd.2026.100310 |