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A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.

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. INTRODUCTIONYellow fever virus (YFV) is a mosquito-borne flavivirus with a present-day distribution confined largely to equatorial Africa and central South America. The enveloped virus contains a single-and positive-stranded RNA genome that encodes a polyprotein of over 350 kDa (Rice et al., 1985;Chambers et al., 1990a) with the following gene order: 59-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-39. C, prM and E are structural proteins and NS1-NS5 are nonstructural proteins involved in polyprotein processing and replication. Numerous studies have shown that the YFV NS3 protease, in complex with NS2B, is involved in coand post-translational processing of the viral polyprotein and recognizes cleavage sequences (G/ARRQS/G) with consensus dibasic residues at the P1 and P2 positions to generate the N termini of NS2B, NS3, NS4A and NS5, and additional cleavage sites within core, NS2A and NS4A (Chambers et al., 1991(Chambers et al., , 1993(Chambers et al., , 1995(Chambers et al., , 2005Amberg et al., 1994;Droll et al., 2000).The YFV NS3 protease belongs to the trypsin superfamily and is relatively well conserved amongst members of the family Flaviviridae (Bazan & Fletterick, 1989;Gorbalenya et al., 1989). The essentiality of NS3 protease activity in viral replication, through at least its polyprotein-cleaving activity, has been demonstrated by mutational analysis of the residues predicted to form the canonical serine protease catalytic triad (H53, D77 and S138) (Chambers et al., 1990b(Chambers et al., , 1993(Chambers et al., , 1995(Chambers et al., , 2005Droll et al., 2000).An effective YFV vaccine was developed when the disease caused havoc in North America at the dawn of the 20th century. There still remains a chance that the disease can re-emerge in outbreak proportion because of its increased incidence in the past 25 years and risks of urban YFV in Africa and South America (Barrett & Higgs, 2007). This has led to calls for the development of chemotherapeutic agents for yellow fever so as not to be caught unp...

Dengue virus (DENV), one of the members of genus Flavivirus is emerging as a global threat to human health. It had led to the emergence of dengue fever (flu-like illness), dengue shock syndrome, and the most severe dengue hemorrhagic fever (severe dengue with bleeding abnormalities). As Dengue hemorrhage diseases are the life-threatening ones, attempts are being made worldwide to design inhibitors for DENV-2 NS2B-NS3 protease. NS2B/NS3 protease plays a vital role in the replication of dengue virus. The trypsin-like serine protease domain of NS3 contains the functional catalytic triad His-51, Asp-75, and Ser-135 in the N-terminal region. Inhibition of the NS3 protease activity is expected to prevent the propagation of dengue virus. Current drug discovery methods are largely inefficient and thus relatively ineffective in tackling the growing threat to public health presented by emerging and remerging viral pathogens. Recently, there has been a need of interest in peptides and their mimetics as potential antagonists for dengue protease because these small peptides are unlikely to invoke an immune response since they fall below the immunogenic threshold. They are often potent and display fewer toxicity issues than small-molecule compounds as a result of high specificity. This study was conducted to design peptides as enzyme inhibitors of dengue virus NS3 protease through computational approach. Crystallographic structure of dengue protease was retrieved from Protein Data Bank (PDBID: 2FOM) and docked with the peptides and the results are analyzed. From the docking studies reported in this paper, tetrapeptide (Lys-Gly-Pro-Glu), pentapeptide (Ser-Ile-Lys-Phe-Ala) and hexapeptide (Ala-Ile-Lys-Lys-Phe-Ser) with glide energy -70.0 kcal/mol, -72.2 kcal/mol and - 80.4 kcal/mol respectively show promising results which can be considered for further optimization and in vitro studies.