Inhibitors of the M2 Proton Channel Engage and Disrupt Transmembrane Networks of Hydrogen-Bonded Waters

Abstract

Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a small-molecule approach is often viewed as insufficient to achieve high affinity and specificity. Here we show that small molecules can enable potent inhibition by targeting key waters. The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine. Structural studies of drug binding to the channel using X-ray crystallography have been limited because of the challenging nature of the target, with the one previously solved crystal structure limited to 3.5 Å resolution. Here we describe crystal structures of amantadine bound to M2 in the Inward conformation (2.00 Å), rimantadine bound to M2 in both the Inward (2.00 Å) and Inward (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inward conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.

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

“…Later, through synthesizing organosilane probes and measuring intermolecular NOESY spectra in DPC micelles, Wang et al (2011a) (Thomaston et al, 2018;. Taken together, these follow-up studies support that amantadine inhibits IAV M2 by plugging the pore ( Figure 5A).…”

“…Regardless, being located at a pore lining residue, the Ser31Asn mutation is likely to alter the diameter as well as the polarity and dynamics of the channel pore, resulting in changes between the observed interactions of the Ser31Asn channel and amantadine when compared to WT M2 (Gleed et al, 2015). Interestingly, in the solved X-ray crystal structure for M2 containing the Ser31Asn mutation (PDB: 5C02; (Thomaston and DeGrado, 2016;Thomaston et al, 2018;, in the absence of a drug molecule in the pore and in the Inward open state, Asn31 was found to face the pore and was stabilized by H-bonds formed with the carbonyl groups of neighboring Asn31…”

1,2-Αnnulated Adamantane Heterocyclic Derivatives as Anti-Influenza Α Virus Agents

“…The hydroxyl of Ser31 forms an internal hydrogen bond to a main-chain carbonyl of Val27, increasing the effective hydrophobicity of the environment. [46] The ammonium group of the Aamt is stabilized due to proximal positioned waters comprising the Ala30 layer, followed by the Gly34 water layer. Placement of the drug within the pore disturbs the overall fourfold rotational symmetry, of the largely symmetrical M2 pore and its water network.…”

“…Thus, Aamts are slightly tilted inside the binding site, with the ammonium group displaced away from the central axis and toward part of the Ala30 waters. [46]…”

“…The all-atom MD simulations were performed with Desmond (Schrodinger, Cambridge, MA). [46,73,74] The POPC lipid bilayer extended 20 Å beyond the solutes in x,y axes, resulting in a system including 120 lipid molecules. The bilayer was solvated using a 20 Å thick layer of TIP3P waters.…”