Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: Influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz–AKRRSQ–EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz–X–Arg–MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister’s series, and significantly influenced by substrate modifications between S4 and S6′. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and kcat, suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.