tailieunhanh - Báo cáo khoa học: NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B–NS3 protease

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. | NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease Xun-Cheng Su1 Kiyoshi Ozawa1 Hiromasa Yagi1 Siew P. Lim2 Daying Wen2 Dariusz Ekonomiuk3 Danzhi Huang3 Thomas H. Keller2 Sebastian Sonntag2 Amedeo Caflisch3 Subhash G. Vasudevan2 and Gottfried Otting1 1 Research Schoolof Chemistry Australian National university Canberra Australia 2 Novartis Institute for TropicalDiseases Singapore 3 Department of Biochemistry University of Zurich Switzerland Keywords drug development inhibitors NMR spectroscopy NS2B-NS3 protease West Nile virus Correspondence G. Otting Research Schoolof Chemistry Australian National university Canberra ACT 0200 Australia Fax 61 2 612 50750 Tel 61 2 612 56507 E-mail go@ Present address Program in Emerging Infectious Diseases Duke-NUS Graduate Medical School Singapore Note Xun-Cheng Su and Kiyoshi Ozawa contributed equally to this work Received 28 February 2009 revised 9 April 2009 accepted 4 June 2009 doi 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 isotopelabelled 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

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