From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus

With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains.

Mechanism and kinetics of copper complexes binding to the influenza A M2 channel

Copper(II) is known to bind in the influenza virus His37 cluster in the homotetrameric M2 proton channel and block the proton current needed for uncoating. Copper complexes based on iminodiacetate also block the M2 proton channel and show reduced cytotoxicity and zebrafish-embryo toxicity. In voltage-clamp oocyte studies using the ubiquitous amantadine-insensitive M2 S31N variant, the current block showed fast and slow phases in contrast to the single phase found for amantadine block of WT M2. Here we evaluate the mechanism of block by copper adamantyl iminodiacitate (Cu(AMT-IDA)) and copper cyclooctyl iminodiacitate (Cu(CO-IDA)) complexes and address whether the complexes can covalently bind to one or more of the His37 imidazoles. The current traces were fitted to parametrized master equations. The energetics of binding and the rate constants suggest that the first step is copper-complex binding within the channel and the slow step in the current block is the covalent bond formation between copper complex and histidine. Isothermal titration calorimetry (ITC) indicates that a single imidazole binds strongly to the copper complexes. Structural optimization using density functional theory (DFT) reveals that the complexes fit inside the channel and project the Cu(II) towards the His37 cluster allowing one imidazole to form a covalent bond with the Cu(II). Electrophysiology and DFT studies also show that the complexes block the G34E amantadine-resistant mutant in spite of some crowding in the binding site by the glutamates.

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

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.