title: NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.
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.
authors: Research School of Chemistry, Australian National University, Canberra, Australia.
authors: Xun-Cheng
authors: XC
authors: Su
authors: Kiyoshi
authors: K
authors: Ozawa
authors: Hiromasa
authors: H
authors: Yagi
authors: Siew P
authors: SP
authors: Lim
authors: Daying
authors: D
authors: Wen
authors: Dariusz
authors: D
authors: Ekonomiuk
authors: Danzhi
authors: D
authors: Huang
authors: Thomas H
authors: TH
authors: Keller
authors: Sebastian
authors: S
authors: Sonntag
authors: Amedeo
authors: A
authors: Caflisch
authors: Subhash G
authors: SG
authors: Vasudevan
authors: Gottfried
authors: G
authors: Otting
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D014902:West Nile virus / Q000187:drug effects / Q000201:enzymology*
chemical_list: C080102:NS2B protein, flavivirus; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases