Yellow fever virus NS3 protease: peptide-inhibition studies

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. 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...

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

“…Although the second least efficiently cleaved amc-labeled substrate, Boc-GRR-amc, showed a 3-fold higher K m (123 µM), turnover was approximately 10-fold greater than for Boc-LRR-amc. In earlier studies Boc-GRR-amc has shown K m of 142 µM and k cat of 0.034 s −1 on NS2B/NS3 protease of YFV and K m of 150 µM and k cat of 0.13 s −1 on NS2B/NS3 protease of DEN2 [34], [35] Thus, this substrate containing P2-P1 Arg-Arg as in cleavage sites of YFV and DEN2 viruses seem to be bound equally tight, but cleaved with lower turnover by JEV protease than by YFV and DEN2 proteases.…”

Molecular targets for flavivirus drug discovery

“…A suitable enzymatic substrate was identified by functional profiling using tetra peptide and octapeptide libraries comprising ~13,000 substrates (Li, J., et al, 2005). Detailed specificity studies have led to the design of robust screening assays in high-throughput formats, employing both colorimetric and fluorescent readouts (Nall et al, 2004; Li, J., et al, 2005; Chappell et al, 2005; 2006; 2007; Gouvea et al, 2007; Knox et al, 2006; Lohr et al, 2007; Radichev et al, 2008; Shiryaev et al, 2006; Shiryaev et al, 2007a, b, c; Yin et al, 2006a,b)…”

Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target

“…Assays with the tetrapeptide substrate Bz-Nle-Lys-Arg-Arg-AMC have been previously described [28]. Briefly, 50 μl reaction mixtures containing 50 mM Tris/HCl, pH 8.5, 1 mM CHAPS, 20% glycerol and 50 μM Bz-Nle-Lys-Arg-Arg-AMC were incubated at 37 • C. Activity was measured in a Victor 3 were determined with plaque assays on BHK-21 cells as previously described [29].…”