The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named “Hv1 Inhibitor Flexibles” (HIFs), consist of two “prongs,” an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.

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