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NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease


Su, X C; Ozawa, K; Yagi, H; Lim, S P; Wen, D; Ekonomiuk, D; Huang, D; Keller, T H; Sonntag, S; Caflisch, A; Vasudevan, S G; Otting, G (2009). NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease. FEBS Journal, 276(15):4244-4255.

Abstract

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2009
Deposited On:03 Nov 2009 08:29
Last Modified:05 Apr 2016 13:31
Publisher:Wiley-Blackwell
ISSN:1742-464X
Additional Information:The definitive version is available at www.blackwell-synergy.com
Publisher DOI:https://doi.org/10.1111/j.1742-4658.2009.07132.x
PubMed ID:19583774
Permanent URL: https://doi.org/10.5167/uzh-23627

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