Molecular Basis for Differential Patterns of Drug Resistance in Influenza N1 and N2 Neuraminidase

Abstract

Neuraminidase (NA) inhibitors are used for the prevention and treatment of influenza A virus infections. Two subtypes of NA, N1 and N2, predominate in viruses that infect humans, but differential patterns of drug resistance have emerged in each subtype despite highly homologous active sites. To understand the molecular basis for the selection of these drug resistance mutations, structural and dynamic analyses on complexes of N1 and N2 NA with substrates and inhibitors were performed. Comparison of dynamic substrate and inhibitor envelopes and interactions at the active site revealed how differential patterns of drug resistance have emerged for specific drug resistance mutations, at residues I222, S246, and H274 in N1 and E119 in N2. Our results show that the differences in intermolecular interactions, especially van der Waals contacts, of the inhibitors versus substrates at the NA active site effectively explain the selection of resistance mutations in the two subtypes. Avoiding such contacts that render inhibitors vulnerable to resistance by better mimicking the dynamics and intermolecular interactions of substrates can lead to the development of novel inhibitors that avoid drug resistance in both subtypes.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes

“…1D) and result in reduced binding affinity between the side pocket and inhibitors, as was observed previously (15,(32)(33)(34). Previously, S246N/G and A246T substitutions were detected in association with NAI resistance in viruses of the N1 and N9 subtypes, respectively (15,32,35). The degree of inhibitor binding of residue 246 was higher for S246 of the N1 subtype than for A246 of the N2 subtype (35).…”

“…The degree of inhibitor binding of residue 246 was higher for S246 of the N1 subtype than for A246 of the N2 subtype (35). Thus, resistance substitutions associated with S246 of the N1 subtype are predominantly detected compared with A246 of the other subtypes (35). However, we have found that an A246T substitution in the N9 subtype and an A246V substitution in the N6 subtype reduced ZA-mediated viral susceptibility.…”

“…Previously, S246N/G and A246T substitutions were detected in association with NAI resistance in viruses of the N1 and N9 subtypes, respectively (15,32,35). The degree of inhibitor binding of residue 246 was higher for S246 of the N1 subtype than for A246 of the N2 subtype (35). Thus, resistance substitutions associated with S246 of the N1 subtype are predominantly detected compared with A246 of the other subtypes (35).…”

Five Novel Non-Sialic Acid-Like Scaffolds Inhibit In Vitro H1N1 and H5N2 Neuraminidase Activity of Influenza a Virus

“…The experiments with the four neuraminidases were carried out in presence of the reference drug zanamivir. The catalytic residues within the active sites were identified as R118, R292 and R371 at subsite S1 and D151 at subsite S2, R152 at subsite S3, and E276 [ 9 , 17 , 36 , 37 ]. They are all interacting with zanamivir whose binding mode allows the drug to tightly attach to the viral enzyme and strongly inhibit the enzymatic activity, since the aforementioned residues are necessary for natural substrate recognition ( Figure 4 ).…”

“…The PDB entries were retrieved from the RCSB Protein Data Bank (RCSB PDB) [ 61 ]. The complete amino acid sequences of the viral neuraminidase were selected according to Prachanronarong [ 36 ] using the Influenza Virus Database ( ) [ 62 ]. The primary sequences were aligned through ClustalW, a web-based multiple sequence alignment tool ( ) [ 63 ].…”