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

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 Inwardclosed conformation (2.00 Å), rimantadine
bound to M2 in both the Inwardclosed (2.00 Å) and
Inwardopen (2.25 Å) conformations, and a spiro-adamantyl
amine inhibitor bound to M2 in the Inwardclosed 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.