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Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR.

ABSTRACT: The M2 protein of influenza A viruses forms a tetrameric proton channel that is targeted by the amantadine class of antiviral drugs. A S31N mutation in the transmembrane (TM) domain of the protein has caused widespread amantadine resistance in most of the currently circulating flu viruses. Recently, a new family of compounds based on amantadine- and aryl-substituted isoxazole were discovered to inhibit the S31N channel activity and reduce replication of S31N-harboring viruses. We now use solid-state NMR spectroscopy to investigate the effects of one of these isoxazole compounds, WJ352, on the conformation of the S31N TM segment and the dynamics of the proton-selective residue, His37. Chemical shift perturbations show that WJ352 changes the conformational equilibrium of multiple TM residues, with the maximal perturbation occurring at the crucial Asn31. (13)C-(2)H distance measurements and (1)H-(1)H NOE cross peaks indicate that the adamantane moiety of the drug is bound in the spacious pore between Asn31 and Gly34 while the phenyl tail is located near Val27. Thus, the polar amine points to the channel exterior rather than to His37, in contrast to amantadine and rimantadine in the wild-type channel, suggesting that the drug is significantly stabilized by hydrophobic interactions between the adamantane and the TM peptide. (15)N and (13)C chemical shifts indicate that at low pH, His37 undergoes fast exchange among the ? tautomer, the ? tautomer, and the cationic state due to proton transfer with water. The exchange rate is higher than the wild-type channel, consistent with the larger single-channel conductance of the mutant. Drug binding at acidic pH largely suppresses this exchange, reverting the histidines to a similar charge distribution as that of the high-pH closed state.

SUBMITTER: Williams JK

PROVIDER: S-EPMC4004037 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR.

Williams Jonathan K JK Tietze Daniel D Wang Jun J Wu Yibing Y DeGrado William F WF Hong Mei M

Journal of the American Chemical Society 20130621 26

The M2 protein of influenza A viruses forms a tetrameric proton channel that is targeted by the amantadine class of antiviral drugs. A S31N mutation in the transmembrane (TM) domain of the protein has caused widespread amantadine resistance in most of the currently circulating flu viruses. Recently, a new family of compounds based on amantadine- and aryl-substituted isoxazole were discovered to inhibit the S31N channel activity and reduce replication of S31N-harboring viruses. We now use solid-s ...[more]

PMID: 23758317

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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.

Dataset Information

Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR.

Publications

Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR.

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