Designing a bis-azide photoaffinity probe in labeling influenza nucleoprotein trimer to give an insight into the binding mode

Abstract

Targeting the highly conserved E330 … R416 salt bridge involved in nucleoprotein (NP) trimerization is considered a favorable approach to address the problem of drug resistance in influenza therapy. A photoaffinity probe (1) containing aryl and alkyl azide moieties was synthesized and applied to investigate its binding mode with influenza NP at the trimeric state. Compound 1 possesses appreciable anti-influenza activity with EC50 ​= ​9.1 ​μM. The photoaffinity labeling experiments were conducted with either the lysate of cells infected by A/WSN/1933(H1N1) virus or the recombinant NP proteins. The aryl azide moiety in bis-azide 1 was selectively activated at 300 ​nm or longer wavelength to enable covalent linkage with the binding site and adjacent amino acid residues. The remaining alkyl azide group was subjected to Cu(I)-catalyzed alkyne–azide cycloaddition with a biotin-tethered alkyne (20), followed by enrichment using streptavidin magnetic beads. Following trypsin digestion, the labeled peptides were identified by LC-MS/MS analysis and Mascot search. Of the five identified NP peptides, four are associated with the NP-trimer binding pocket. Molecular docking experiments show that the 2-anilino-1,3-thiazole-4-carboxamide backbone of compound 1 occupies almost the same position of E339 … R416 salt bridge to form hydrogen bonds with E339, providing evidence for interference with NP trimerization.

Keywords

• Influenza
• Nucleoprotein
• Photoaffinity
• Bis-azide
• LC-MS/MS
• Molecular docking