Chemical Probes for Blocking of Influenza A M2 WT and S31N Channels

We report on using the synthetic aminoadamantane-CH 2 -aryl derivatives 1–6 as sensitive probes for blocking M2 S31N and M2 WT channels as well as virus replication in cell culture. The binding kinetics for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl head group realized in 2 and 3 , and the girth and length of the adamantane adduct realized in 4 and 5 . Study of 1–6 show that, according to MD simulations and 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 as well as triamantane insetad of adamantane in 4 and 5 , respectively, causes an 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, corresponding to high k on / low k off and high k on / high k off measured rate constants, compared to inactive 2–5 , which have much lower k on and higher k off . Compounds 1–5 each block the M2 WT channel by binding in the longer area from V27 - H37, 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 1 – 5 and 1 – 4 , and 6 respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2–4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.