Chemical Probes for Blocking of Influenza A M2 Wild-type and S31N Channels

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 16 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 16 shows that, according to molecular dynamics (MD) simulations and molecular mechanics PoissonBoltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high k(on)/low k(off) and high k(on)/high k(off) rate constants, compared to inactive 2-5, which have much lower k(on) and higher k(off). Compounds 15 block the M2 WT channel by binding in the longer area from V27H37, in the inward orientation, with high k(on) and low k(off) rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 15 or 14 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 24 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.