Unraveling the Binding, Proton Blockage, and Inhibition of Influenza M2 WT and S31N by Rimantadine Variants

Abstract

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher rates compared to the rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding and against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.

Rotation Barriers of 1‐Adamantyl‐Csp3 Bonds Measured with Dynamic NMR

“…We were intrigued to further investigate the behavior of the 1‐adamantyl group in rotation barriers using DNMR, following up our previous relevant studies . The tertiary alcohol AdCEt 2 OH ( 11 ) was prepared in high yield from the reaction between 1‐adamantane carbonyl chloride and ethyl lithium, as has been previously described and fully characterized . AdCEt 2 Cl (12 ) was prepared from reaction of 11 with HCl(g) in anhydrous CH 2 Cl 2 (‐50 °C, 15 min) .…”

(2-Hydroxy-4-methoxy)benzyl Aminoadamantane Conjugates as Probes to Investigate Specificity Determinants in Blocking Influenza M2 S31N and M2 WT Channels with Binding Kinetics and Simulations

“…WT and M2 V27A. 36,37 We synthesized compounds 2-5 as blockers of both M2 S31N and WT proton channels after biomolecular simulations sugggested them as competent binders. It was unexpected that although 1-5 efficiently block M2 WT, they do not block M2 S31N, which leads to the observation that even the small linker CMe 2 in 2 abolishes M2 S31N blocking.…”

“…The percent-washout and k off are similar to those of compounds 3 and 4, but the k on is very high and complete (99.2%) block is attained, such that 6 has high potency in blocking M2 S31N, as well as in blocking virus strains replication containing this amantadine-insensitive mutation. 3 Interestingly, k on for 6 in blocking M2 S31N is dramatically higher than others tested to date [3][4][5][6][7][8][9][10]37 ( Table 4). Yet, the rate constant is still 6-7 orders of magnitude lower than expected for unhindered diffusion-limited binding, 43,44 suggesting a substantial energetic barrier or kinetic limitation along the reaction coordinate from bulk solution to the current-blocking site.…”

“…Using two-electrode voltage clamp (TEVC) we calculated %-blocking at 10 min of drug perfusion (Table 3) to obtain more reliable results, as we recently suggested, 23,36,37 compared to 2 min usually determined in studies dealing with testing of new drug molecules from synthesis. [4][5][6]10,38 we found that molecules 1-5 block strongly M2 WT-mediated proton.…”

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

“…Combination therapy for drug-resistant influenza M2 compared to other minor variants [23][24][25][26][27][28][29][30][31]. The majority of studies have focused upon adamantane derivatives with various chemical groups linked via the primary amine.…”