Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B–NS3 proteases

[Display omitted] •We designed and evaluated peptide library against the proteases of dengue viruses.•Substrate based octapeptide inhibitors with low μM activity were identified.•Shortening and modifications resulted in uncharged tetrapeptide inhibitor.•Activity data analysis and docking gave insights in peptide–protease interactions. A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B–NS3 proteases of the four subtypes of dengue virus, DEN-1–4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2μM for the DEN-1–4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.