X-ray Crystal Structures of the Influenza M2 Proton Channel Drug-Resistant V27A Mutant Bound to a Spiro-Adamantyl Amine Inhibitor Reveal the Mechanism of Adamantane Resistance

Abstract

The V27A mutation confers adamantane resistance to the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to solve structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inward closed conformation. The spiroadamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in both the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics (MD) simulation of *

Put a cork in it: Plugging the M2 viral ion channel to sink influenza

“…-S31N (19-49) in complex with (4) (PDB: 2LY0; ). (C) X-ray crystal structure of M2-V27A (22-46) in complex with spiro-adamantyl amine (9) (PDB: 6NV1; Thomaston et al, 2020).…”

“…Among these, Ser31Asn is the prevalent mutation in more than 95% of adamantane-resistant strains (Dong et al, 2015;Nelson et al, 2009), although a recent study reported a potential rise in the frequency of virus strains containing the Val27Ala and Ser31Asn double mutations (Durrant et al, 2014). A recent crystallography study reported that elimination of hydrophobic contacts that are essential for adamantane drug binding is the underlying cause behind adamantane resistance to Val27Ala mutant (Thomaston et al, 2020). In contrast to the Val27Ala single mutation that results in complete resistance to amantadine, M2 sequences containing Ser31Asn can still be inhibited by amantadine in vitro, albeit with approximately 200-fold higher concentrations in TEVC experiments and plaque reduction assays (PRAs) and substantially higher than what is therapeutically-achievable (Wang et al, 2013a).…”

Anti-Influenza Drug Discovery and Development: Targeting the Virus and Its Host by All Possible Means

“…Upon drug-resistance V27A mutation, this interaction is lost. Recentlydeveloped spiro-amantadyl amine effectively binds to A27 of the pore (Figure 2B) [136]. Recently, new amantadine derivatives effective against double mutants M2-S31N/L26I and M2-S31N/V27A viral strains have been developed by Musharrafieh et al [91].…”

Amantadine Variants Activity Against Multiple Influenza A Viruses

“…3 Several groups published high resolution structures and data from mutation experiments showing that M2 protein channel is blocked by 1 and 2 via a M2 pore-binding mechanism. [4][5][6][7][8][9] Additionally, amantadine variants, especially those with hydrophobic adducts, can act as lysosomotropic drugs, 10 which accumulate in intracellular vesicles through membrane permeation by the electroneutral form and increase intravesicular pH, causing endosome and/or trans-Golgi network neutralization and inhibition of viral reproduction. 11 Noteworthy, SARS-CoV-2 is inhibited by chloroquine, probably because it acts as a lysosomotropic drug.…”