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Mol Pharmacol. 2019 Jun 7. pii: mol.119.116640. doi: 10.1124/mol.119.116640. [Epub ahead of print]

The L46P mutant confers a novel allosteric mechanism of resistance towards the influenza A virus M2 S31N proton channel blockers.

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University of Arizona.
National and Kapodistrian University of Athens.
J Craig Venter Institute.
University of Arizona;


The FDA-approved influenza A antiviral amantadine inhibits the wild-type (WT) AM2 channel but not the S31N mutant predominantly found in circulating strains. In this study, serial viral passages were applied to select resistance against a newly developed isoxazole-conjugated adamantane inhibitor that targets the AM2 S31N channel. This led to the identification of the novel drug-resistant mutation L46P located outside the drug binding site, which suggests an allosteric resistance mechanism. Intriguingly, when the L46P mutant was introduced to AM2 WT, the channel remained sensitive towards amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics (MD) simulations and binding free energy molecular mechanics-generalized born surface area (MM-GBSA) calculations were performed on WT and mutant channels. It was found that the L46P mutation caused a conformational change in the N-terminus of the transmembrane residues 22-31 that ultimately broadened the drug binding site of AM2 S31N inhibitor 4, which spans residues 26-34, but not of AM2 WT inhibitor amantadine, which spans residues 31-34. The MM-GBSA calculations showed stronger binding stability for 4 in complex with AM2 S31N compared to its complex with AM2 S31N/L46P, and equal binding free energies of amantadine in complex with AM2 WT and AM2 L46P. Overall, these results demonstrate a unique allosteric resistance mechanism towards AM2 S31N channel blockers, and the L46P mutant represents the first experimentally confirmed drug-resistant AM2 mutant that is located outside of the pore where drug binds. SIGNIFICANCE STATEMENT: AM2 S31N is a high profile antiviral drug target as more than 95% of current circulating influenza A viruses carry this mutation. Understanding the mechanism of drug resistance is critical in designing the next generation of AM2 S31N channel blockers. Using one of our previously developed AM2 S31N channel blocker as a chemical probe, we, for the first time, identified a novel resistant mutant L46P. The L46P mutant located outside of the drug binding site. It was shown by molecular dynamics simulations that the L46P causes a dilation of drug binding site between residues 22-31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor, amantadine.


Antiviral drugs; Electrophysiology; Ion channels

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