Discovery of M2 channel blockers targeting the drug-resistant double mutants M2-S31N/L26I and M2-S31N/V27A from the influenza A viruses

Influenza virus infections are a persistent threat to human health due to seasonal outbreaks and sporadic pandemics. Amantadine and rimantadine are FDA-approved influenza antiviral drugs and work by inhibiting the viral M2 proton channel. However, the therapeutic potential for the antiviral amantadine/rimantadine was curtailed by the emergence of drug-resistant mutations in its target protein M2. In this study, we identified four amantadine-resistant M2 mutants among avian and human influenza A H5N1 strains circulating between 2002 and 2019: the single S31N and V27A mutants, and the S31N/L26I and S31N/V27A double mutants. Herein, utilizing two-electrode voltage clamp (TEVC) assays, we screened a panel of structurally diverse M2 inhibitors against these single and double mutant channels. Three compounds 6, 7, and 15 were found to significantly block all three M2 mutants: M2-S31N, M2-S31N/L26I, and M2-S31N/V27A. Using recombinant viruses generated from reverse genetics, we further showed that these compounds also inhibited the replication of recombinant viruses harboring either the single S31N or double S31N/L26I and S31N/V27A mutants. This work represents the first example in developing antivirals by targeting the drug-resistant double mutants of M2 proton channels.