The NS3 serine protease of dengue virus is required for the maturation of the viral polyprotein and consequently represents a promising target for the development of antiviral inhibitors. However, the substrate specificity of this enzyme has been characterized only to a limited extent. In this study, we have investigated product inhibition of the NS3 protease by synthetic peptides derived from the P6-P1 and the P1'-P5' regions of the natural polyprotein substrate. N-terminal cleavage site peptides corresponding to the P6-P1 region of the polyprotein were found to act as competitive inhibitors of the enzyme with K-i values ranging from 67 to 12 mu M. The lowest K-i value was found for the peptide representing the NS2A/NS2B cleavage site, RTSKKR. Inhibition by this cleavage site sequence vias analyzed by using shorter peptides, SKKR, KKR, KR, AGRR, and GKR. With the exception of the peptide AGRR which did not inhibit the protease at a concentration of 1 mM, all other peptides displayed Ki values in the range from 188 to 22 mu M. Peptides corresponding to the P1'-P5' region of the polyprotein cleavage sites had no effect on enzymatic activity at a concentration of 1 mM. Molecular docking data of peptide inhibitors to a homology-based model of the dengue virus type 2 NS2B(H)-NS3p co-complex indicate that binding of the non-prime site product inhibitors is similar to ground-state binding of the corresponding substrates. (c) 2005 Elsevier Inc. All rights reserved.