Studying the Effect of Amino Acid Substitutions in the M2 Ion Channel of the Influenza Virus on the Antiviral Activity of the Aminoadamantane DerivativeIn VitroandIn Silico

Abstract

Purpose: The aminoadamantane derivative of L-histidyl-1-adamantayl ethylamine hydrochloride (HCl*H-His-Rim) has showed a high inhibition level against influenza A virus strains in vitro. The aim of this work is to search and establish evidence of the direct effect of the drug on influenza A virus proton channel M2. Methods: The compound HCl*H-His-Rim was obtained by classical peptide synthesis methods. Influenza A virus mutants of A/PuertoRico/8/34(H1N1) strain were obtained by reverse genetics methods. The mutant samples of the virus were cultured on chicken embryos with a virus titer in the hemagglutination test. ELISA was carried out on Madin-Darby canine kidney (MDCK) monolayer cells when multiplying the virus 10-4-10-6. The binding stability of HCl*H-His-Rim was compared to those of M2 (S31N) and M2 (S31N_A30T) channels by molecular dynamic (MD) modeling. The calculation was performed taking into account the interaction with the model lipid bilayer (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) in the presence of water molecules in accordance with the three-center model. Results: It was found that HCl*H-His-Rim is a direct action drug against influenza A. The most likely conformation of drug binding to target protein has been shown. It has been found that the A30T mutation reduces the binding energy of the drug, and the results obtained in vitro have confirmed the data calculated in silico. Conclusion: The mechanism of action of HCl*H-His-Rim is directly related to the suppression of the function of the proton channel M2 of influenza A virus.

Alchemical Free Energy Calculations on Membrane-Associated Proteins

“…In other studies on the M2 proton channel of influenza A, RBFE calculations were used to investigate the change in the affinity of ( R )- versus ( S )- enantiomers of the anti-influenza drug rimantadine. , In agreement with electrophysiology kinetic assays, the FEP results showed no difference in binding affinity, supporting the conclusion that the chirality of drugs binding the M2 proton channel has no effect on binding or channel inhibition despite the M2 pore being chiral. In another investigation on the proton channel M2, absolute FEP calculations were used to predict the binding site of an aminoadamantane derivative known to produce strong inhibition against influenza virus A in vitro . The computational predictions validated on two experimentally characterized protein mutants suggest that this aminoadamantane derivative deeply penetrates the M2 channel pore strengthening the hypothesis that its antiviral effect derives from an open-channel block .…”

“…In another investigation on the proton channel M2, absolute FEP calculations were used to predict the binding site of an aminoadamantane derivative known to produce strong inhibition against influenza virus A in vitro . The computational predictions validated on two experimentally characterized protein mutants suggest that this aminoadamantane derivative deeply penetrates the M2 channel pore strengthening the hypothesis that its antiviral effect derives from an open-channel block . The same approach was used to refine docking poses of the general anesthetic sevofluorane on the voltage-gated potassium channel Kv1.2 and obtain a 3D map highlighting the complexity of molecular recognition at membrane receptors with multiple binding sites with single or multiple occupancy states being in competition. , Last, both absolute and relative FEP calculations in ion channels were used to assign ambiguous disconnected electron densities in protein crystal structures.…”

Physiologically Active Compounds Based on Membranotropic Cage Carriers–Derivatives of Adamantane and Polyhedral Boron Clusters (Review)

“…For the compound ( Fig. 5a ), the mechanism of action in silico and in vitro was determined by comparing the results of molecular docking and antiviral properties of artificially created mutants of the A/PuertoRico/8/34 (H1N1) virus with point amino acid substitutions in the transmembrane region of the M2 protein [ 31 ].…”