STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

Autor: Schöne T; Center for Structural and Cell Biology in Medicine, Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel Site, University of Lübeck, Germany., Grimm LL; Center for Structural and Cell Biology in Medicine, Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany., Sakai N; Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany., Zhang L; Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany., Hilgenfeld R; Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel Site, University of Lübeck, Germany., Peters T; Center for Structural and Cell Biology in Medicine, Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany. Electronic address: thomas.peters@chemie.uni-luebeck.de.
Jazyk: angličtina
Zdroj: Antiviral research [Antiviral Res] 2017 Oct; Vol. 146, pp. 174-183. Date of Electronic Publication: 2017 Sep 18.
DOI: 10.1016/j.antiviral.2017.09.008
Abstrakt: West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors.
(Copyright © 2017 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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