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Investigation of the Drug Resistance Mechanism of M2-S31N Channel Blockers through Biomolecular Simulations and Viral Passage Experiments.

ABSTRACT: Recent efforts in drug development against influenza A virus (IAV) M2 proton channel S31N mutant resulted in conjugates of amantadine linked with aryl head heterocycles. To understand the mechanism of drug resistance, we chose a representative M2-S31N inhibitor, compound 3, as a chemical probe to identify resistant mutants. To increase the possibility of identifying novel resistant mutants, serial viral passage experiments were performed with multiple strains of H1N1 and H3N2 viruses in different cell lines. This approach not only identified M2 mutations around the drug-binding site, including the pore-lining residues (V27A, V27F, N31S, and G34E) and an interhelical residue (I32N), but also a new allosteric mutation (R45H), in addition to L46P previously identified, located at the C-terminus of M2 that is more than 10 Å away from the drug-binding site. The effects of each mutation were next investigated using electrophysiology, recombinant viruses, and molecular dynamics (MD) simulations. The reduced sensitivity in channel blockage correlated with increased drug resistance in antiviral assays using recombinant viruses. The MD simulations show that the V27A, V27F, G34E, and R45H mutations increase the diameter and hydration state of the pore in complex with compound 3. The Molecular Mechanics Generalized Born (MM-GBSA) calculations result in more positive binding free energies for the complexes of resistant M2 (V27A, V27F, G34E, R45H) with compound 3 compared to the stable complexes (S31N and I32N). Overall, this is the first systematic study of the drug resistance mechanism of M2-S31N channel blockers using multiple viruses in different cell lines.

SUBMITTER: Musharrafieh R

PROVIDER: S-EPMC7432665 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Food and Drug Administration-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 toward amantadine inhibition. To elucidate the molecular mechanism, molecular dynamics 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 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 with 4 in 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 toward 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 currently 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 a previously developed AM2 S31N channel blocker as a chemical probe, this study was the first to identify a novel resistant mutant, L46P. The L46P mutant is located outside of the drug-binding site. Molecular dynamics simulations showed that L46P causes a dilation of drug-binding site between residues 22 and 31, which affects the binding of AM2 S31N channel blockers, but not the AM2 WT inhibitor amantadine.

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Investigation of the Drug Resistance Mechanism of M2-S31N Channel Blockers through Biomolecular Simulations and Viral Passage Experiments.

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