Exploiting Π-Stacking Interactions to Improve Inhibition of the Hv1 Channel by Aromatic Guanidine Derivatives

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The human Hv1 voltage-gated proton channel is an emerging drug target for cancer and stroke. It resides on the cell plasma membrane and endocytic compartments where it mediates outward proton movement and regulates the activity of NOX enzymes. The channel consists of two identical subunits which gate cooperatively. Each subunit contains a proton-conducting voltage-sensing domain (VSD) which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). We have previously found that mutating the Hv1 residue F150 to an alanine increases the 2GBI binding affinity more than two orders of magnitude. Understanding how 2GBI and its analogs interact with Hv1 F150A is critical to the design of more effective inhibitors for the wild type channel. We hypothesized that the binding of 2GBI to the mutant channel is stabilized by π-stacking interactions between the inhibitor and aromatic residues located in the core of the VSD. We tested this hypothesis using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated aromatic amino acids via nonsense suppression methodology. Considering their proximity to the 2GBI binding site, we investigated residues F149 and F182. We found that F182 does contribute to 2GBI binding via π-stacking interactions while F149 does not. To test whether strengthening the π-stacking interactions can lead to more potent inhibitors, we measured the binding affinities of a series of aromatic guanidine derivatives carrying an increasing number of fluorine substituents. We discuss the results based on our understanding of the mechanism of channel block by these compounds. Possible alterations of the inhibitor structure to improve binding to the wild type channel are also discussed.