Antiviral Mechanisms and Preclinical Evaluation of Amantadine Analogs that Continue to Inhibit Influenza A Viruses with M2 S31N-Based Drug Resistance

To better manage seasonal and pandemic influenza infections, new drugs are needed with enhanced activity against contemporary amantadine- and rimantadine-resistant influenza A virus (IAV) strains containing the S31N variant of the viral M2 ion channel (M2S31N). Here we tested 36 amantadine analogs against a panel of viruses containing either M2S31N or the parental, M2 S31 wild-type variant (M2WT). We found that several analogs, primarily those with sizeable lipophilic adducts, inhibited up to three M2S31N-containing viruses with activities at least 5-fold lower than rimantadine, without inhibiting M2S31N proton currents or modulating endosomal pH. While M2WT viruses in passaging studies rapidly gained resistance to these analogs through the established M2 mutations V27A and/or A30T, resistance development was markedly slower for M2S31N viruses and did not associate with additional M2 mutations. Instead, a subset of analogs, exemplified by 2-propyl-2-adamantanamine (38), but not 2-(1-adamantyl)piperidine (26), spiro[adamantane-2,2-pyrrolidine] (49), or spiro[adamantane-2,2-piperidine] (60), inhibited cellular entry of infectious IAV following pre-treatment and/or H1N1 pseudovirus entry. Conversely, an overlapping subset of the most lipophilic analogs including compounds 26, 49, 60, and others, disrupted viral M2-M1 protein colocalization required for intracellular viral assembly and budding. Finally, a pilot toxicity study in mice demonstrated that 38 and 49 were tolerated at doses approaching those of amantadine. Together, these results indicate that amantadine analogs act on multiple, complementary mechanisms to inhibit replication of M2S31N viruses.