Increased Dissociation of Adamantanamines in Influenza A M2 S31N with Partial Block by Rimantadine

Area of Science:

• Virology
• Molecular Biology
• Biophysics

Background:

• Influenza A M2 channel mutations, like S31N, confer resistance to antiviral drugs amantadine (AMT) and rimantadine (RMT).
• The precise mechanism of resistance, whether due to weak binding or incomplete channel block, remains unclear.

Purpose of the Study:

• To elucidate the mechanism by which the S31N mutation affects amantadine and rimantadine inhibition of the influenza A M2 channel.
• To differentiate between weak binding and incomplete block as causes of drug resistance.

Main Methods:

• Two-electrode voltage clamp (TEVC) electrophysiology on Xenopus oocytes expressing mutant M2 channels.
• Kinetic analysis of proton current wash-in and wash-out traces to determine rate constants (k1, k2).
• Adaptive biasing force (ABF) molecular dynamics simulations to calculate potentials of mean force (PMF).

Main Results:

• Amantadine (AMT) binding to the S31N M2 channel, though weak, results in complete block of proton current.
• Rimantadine (RMT) exhibits only modest block of the S31N M2 channel, even at high concentrations, indicating binding site saturation.
• Rate constant analysis revealed decreased association (k1) and dramatically increased dissociation (k2) for both drugs in the S31N mutant.
• Molecular dynamics simulations correlated rate constants with the charge state of the His37 residue in the M2 channel's selectivity filter.

Conclusions:

• The S31N mutation confers amantadine resistance through weak binding that still achieves complete proton channel block.
• Rimantadine resistance is characterized by binding site saturation leading to incomplete block.
• Molecular dynamics simulations support experimental findings and highlight the role of the His37 charge state in drug interactions.