Influenza a Blockers with Reduced Resistance Formation

Influenza A develops amantadine resistance within days in tissue culture or infection. The amantadine-resistant mutant, M2 S31N, has become globally dominant in human isolates. Infection of MDCK cells by a pandemic 2009 H1N1 strain, bearing M2 S31N, was blocked by low-micromolar concentrations for a set of amantadine analogs previously shown to block several M2-WT influenza strains from H1N1, H2N2, and H3N2 subtypes. Three sets of variants were synthesized to explore SAR properties. One demonstrated that opening the adamantane cage reduced activity. Another showed that the adduct must be more than 2 carbons. Pre-exposing the virus to drug before inoculation showed inactivation and exposure-recovery of the virus on the ∼10-minute time scale. Resistance development is dramatically reduced. For selected compounds, 10 passages (∼5 weeks) in the presence of drug were required before the 2009 H1N1 developed resistance. However, the mechanism of action is unclear. Liposome assays indicate direct block of S31N M2 (22-62). But 2009 H1N1 M2-transfected HEK cells are not blocked, either on the 2- or the 30-minute time scales. Yet, the revertant (N31S) is well blocked. Solid state NMR suggests that drugs bind to the S31N transmembrane peptide domain. The resistant strains developed in the presence of these drugs show no mutations in M2, but a few mutations in hemagglutinin. It is possible that these hydrophobic amines function partly by neutralizing the endosome. However, the virus pre-exposure results indicate a direct effect on the virus, not just on the endosome. The A/WSN/33 virus is not blocked by these drugs in cytopathic effect assays, but the revertant (N31S) is, indicating for A/WSN/33 that the M2-block is the key effect. In summary, resistance-invulnerable drugs for the 2009 H1N1 influenza A virus have been identified and the mechanism of action is yet to be defined.