ABSTRACT
A recombinant form of yellow fever virus (YFV) NS3 protease, linked via a nonapeptide to the minimal NS2B co-factor sequence (CF40-gly-NS3pro190), was expressed in Escherichia coli and shown to be catalytically active. It efficiently cleaved the fluorogenic tetrapeptide substrate Bz-norleucine-lysine-arginine-arginine-AMC, which was previously optimized for dengue virus NS2B/3 protease. A series of small peptidic inhibitors based on this substrate sequence readily inhibited its enzymic activity. To understand the structure-activity relationship of the inhibitors, they were docked into a homology model of the YFV NS2B/NS3 protease structure. The results revealed that the P1 and P2 positions are most important for inhibitor binding, whilst the P3 and P4 positions have much less effect. These findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.
Subject(s)
Enzyme Inhibitors/pharmacology , Oligopeptides/metabolism , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/metabolism , Yellow fever virus/enzymology , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites/physiology , Kinetics , Molecular Sequence Data , Oligopeptides/chemistry , RNA Helicases/chemistry , RNA Helicases/drug effects , RNA Helicases/genetics , RNA Helicases/metabolism , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Sequence Alignment , Serine Endopeptidases/chemistry , Serine Endopeptidases/drug effects , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Substrate Specificity , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.
