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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: 3.53). 09/2007; 88(Pt 8):2223-7. DOI: 10.1099/vir.0.82735-0
Source: PubMed

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.

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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
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    • "The assay has been described previously [14]. Briefly, activities of cNS2B/NS3pro complexes were measured in a Safire 2 plate reader (Tecan) (k ex = 385 nM, k em = 465 nM) and performed in a final volume of 50 ll containing 50 mM Tris–HCl (pH 7.5), 1 mM Chaps, 20% glycerol, and 50 lM Bz-Nle-Lys-Arg-Arg-AMC at 37 °C. "
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    ABSTRACT: In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.
    Analytical Biochemistry 09/2009; 395(2):195-204. DOI:10.1016/j.ab.2009.08.013
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    • "We believe this knowledge will be highly valuable for the precise understanding of the NS2B–NS3pro molecular complex formation and for the optimization of the selective allosteric small-molecule inhibitors designed to target the NS2B–NS3pro interface. We suspect that these allosteric inhibitors designed to inactivate the twocomponent flaviviral proteinase by interfering with the NS2B–NS3pro interactions, will not cross-react with host cell serine proteinases and, accordingly, they will exhibit fewer side effects when compared with the active sitetargeting antagonists of the flaviviral NS2B–NS3pro (Knox et al., 2006; Lohr et al., 2007; Yin et al., 2006). "
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    ABSTRACT: West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B-NS3pro structure, we hypothesized that the G(22) residue and the negatively charged patch D(32)DD(34) of NS2B were part of an important configuration required for NS2B-NS3pro activity. Our experimental data confirmed that G(22) and D(32)DD(34) substitution for S and AAA, respectively, inactivated NS2B-NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B-NS3pro. Because of the significant level of homology in flaviviral NS2B-NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.
    Journal of General Virology 04/2008; 89(Pt 3):636-41. DOI:10.1099/vir.0.83359-0
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