Novel tools to study West Nile virus NS3 protease activity.

West Nile Virus (WNV) belongs to a group of pathogenic viruses called flaviviruses. West Nile virus infection can be mild, causing so-called West Nile Fever (WNF) or severe neuroinvasive form of the disease (WNND), and ultimately even death. There are currently no known medications to prevent West Nile virus infection. Only symptomatic treatment is used. To date, there are no unequivocal tests enabling a quick and unambiguous assessment of WN virus infection. The aim of the research was to obtain specific and selective tools for determining the activity of the West Nile virus serine proteinase. Using the methods of combinatorial chemistry with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. The FRET ABZ-Ala-Lys-Gln-Arg-Gly-Gly-Thr-Tyr(3-NO2)-NH2 substrate was obtained, characterized by kinetic parameters (KM = 4.20 ± 0.32 × 10-5 M) as for the majority of proteolytic enzymes. The obtained sequence was used to develop and synthesize highly sensitive functionalized quantum dot-based protease probes (QD). A QD WNV NS3 protease probe was obtained to detect an increase in fluorescence of 0.05 nmol enzyme in the assay system. This value was at least 20 times lower than that observed with the optimized substrate. The obtained result may be the basis for further research on the potential use of the WNV NS3 protease in the diagnosis of West Nile virus infection.