Yellow fever virus NS3 protease: Peptide-inhibition studies

Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670.
Journal of General Virology (Impact Factor: 3.18). 09/2007; 88(Pt 8):2223-7. DOI: 10.1099/vir.0.82735-0
Source: PubMed


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.

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    • "c o m / l o c a t e / y b b r c [10]. Non-prime side peptide sequences, and compounds based on aromatic scaffolds were also found, which inhibited NS2B–NS3 proteases of West Nile and yellow fever viruses [11] [12] [13] [14]. "
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    ABSTRACT: A series of forty-five 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 11.2 μM 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.
    Biochemical and Biophysical Research Communications 04/2013; DOI:10.1016/j.bbrc.2013.03.139 · 2.30 Impact Factor
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    • "Although the second least efficiently cleaved amc-labeled substrate, Boc-GRR-amc, showed a 3-fold higher Km (123 µM), turnover was approximately 10-fold greater than for Boc-LRR-amc. In earlier studies Boc-GRR-amc has shown Km of 142 µM and kcat of 0.034 s−1 on NS2B/NS3 protease of YFV and Km of 150 µM and kcat 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. "
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    ABSTRACT: Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20-30% are fatal, while 30-50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K(m) and k(cat), for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)(4)SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k(cat)/K(m) was found for Pyr-RTKR-amc (k(cat)/K(m): 1962.96 ± 85.0 M(-1) s(-1)) and the lowest for Boc-LRR-amc (k(cat)/K(m): 3.74±0.3 M(-1) s(-1)). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.
    PLoS ONE 05/2012; 7(5):e36872. DOI:10.1371/journal.pone.0036872 · 3.23 Impact Factor
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    • "Recombinant YFV NS2B/NS3 protease was obtained and purified as previously described by [22]. Briefly, the 47 core amino acids of NS2B were linked (via Gly4-Ser-Gly4) to the N-terminally 190 amino acids of NS3 protease domain [18] and expressed in E. coli M15 strain as an N-terminally his-tagged fusion protein. "
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    ABSTRACT: Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P'1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k(cat)/K(m) of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity - a unique feature among worldwide prominent flavivirus - was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k(cat)/K(m). The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates.
    Biochemical and Biophysical Research Communications 03/2011; 407(4):640-4. DOI:10.1016/j.bbrc.2011.03.054 · 2.30 Impact Factor
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