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 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.