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

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

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

Antiviral Res. 2017 Sep 16;:

Authors: Schöne T, Grimm LL, Sakai N, Zhang L, Hilgenfeld R, Peters T

Abstract
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 (IC50 ~ 500*?M) 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.


PMID: 28927677 [PubMed - as supplied by publisher]



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