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• W98993685 abstract "West Nile Virus (WNV) is an emerging pathogen with an expanding geographic distribution. Over the last decade it has been the cause of an increasing frequency of human outbreaks with associated severe human disease and fatalities. Currently, there is no approved vaccine or effective antiviral strategy available for combating WNV. One attractive target for antiviral development is a viral, trypsin-like, serine protease, which is encoded by the N-terminal 184 amino acids of NS3 and is only active as a heterodimeric complex with its cofactor, NS2B. This protease, NS2B/NS3pro, plays an essential role in the cleavage of the viral precursor polyprotein and disruption of this function has been shown to be lethal for virus replication. In this thesis I have examined the structure and function of WNV NS2B/NS3pro by using a recombinant WNV protease, which we previously generated (Nall et al., 2004), and a combination of approaches including site-directed mutagenesis, truncation studies and the screening of small libraries of peptide substrates and inhibitors. The results obtained have made a significant contribution to the fundamental understanding of the NS2B/NS3protease and the residues involved in substrate-binding, resulting in three international publications. The first publication, Chappell et al., (2005), comprises a mutagenesis study directed to the substrate-binding cleft, which was undertaken prior the resolution of the active crystal structure of the WNV protease by Erbel et al., (2006). Mutagenesis results were interpreted based on a homology model of WNV protease. While some of the results that were obtained are highly intuitive and provide an understanding of substrate binding, others have now been re-evaluated in the context of the crystal structure of the active protease and found to be located far from the substrate-binding cleft. This re-evaluation of the mutagenesis results is included in information supplementary to this publication. The second publication, Chappell et al., (2006a), encompasses a more comprehensive analysis of the substrate-binding cleft, which was undertaken after the resolution of the crystal structure of the active protease. This study uses both site-directed mutagenesis and screening of a small library of peptide substrates to investigate substrate binding and the results are interpreted by docking of substrates into the active crystal structure of WNV NS2B/NS3pro. The substrate specificity at the P2, P3 and P4 positions was thoroughly investigated as well as the individual residues of the NS3 protease and the NS2B cofactor involved in substrate-binding interactions. One particular residue within the cofactor was found to strongly influence binding preference of Flavivirus proteases for an arginine or lysine residue at P2. The results of this study provide an overview of substrate binding, in which the substrate is predicted to bind in an extended conformation, as opposed to that crystallographically demonstrated for a small inhibitor bound in the active site of WNV NS2B/NS3pro. The identified substrate-binding residues and the optimal tetra-peptide substrate provide a base for rational drug design of protease inhibitors. The development and analysis of aldehyde inhibitors based on the identified optimal substrate is discussed in the supplementary material. The third publication, Chappell et al., (2006b) focused on truncation and optimisation of the previously generated recombinant protease. This resulted in the generation of a highly stable construct which can be used in future studies including inhibitor screening and crystallisation trials. This construct has now been successfully crystallised in association with a potent aldehyde inhibitor and the resolution of this structure in the near future will provide valuable information for further inhibitor development. A stable recombinant protease incorporating the full length of NS3 was also generated and a comparison of the enzyme kinetics of the two recombinant constructs reveals that the C-terminal domain has only a small effect on protease activity. The information presented in this thesis provides a base for rational drug design of inhibitors of the WNV protease and has so far led to the development of aldehyde inhibitors which constitute the most highly potent, small peptide inhibitors identified to date. These lead inhibitors are suitable for future rational drug design and structural optimization. This has the potential to lead to the generation of broad spectrum antiviral drug candidates for the treatment of infections by WNV and other Flaviviruses." @default.
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• W98993685 date "2007-01-01" @default.
• W98993685 modified "2023-09-26" @default.
• W98993685 title "Structure-function relationships of the West Nile Virus protease NS3 and its cofactor NS2B" @default.
• W98993685 hasPublicationYear "2007" @default.
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