Two-Pronged Aromatic Arginine-Mimics as Hv1 Proton Channel Inhibitors

Abstract

The voltage-gated proton channel Hv1 regulates cellular pH homeostasis and the production of reactive oxygen species by the NADPH oxidase. The channel is made of two identical subunits, which gate cooperatively. Each subunit contains a voltage-sensing domain (VSD) that conducts protons and is inhibited by the arginine mimic 2-guanidinobenzimidazole (2GBI). 2GBI operates from the intracellular side and gains access to its binding site when the channel is open. The blocker must leave the binding site for the channel gate to close. We previously found that mutation F150A strongly increases the binding affinity. We now show that this increase in affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150 and use this information to design new arginine mimics with improved affinity for the non-rearranged binding site of the wild type channel. The new compounds consist of two “prongs”, an aminoimidazole ring and a fluorinated aromatic group connected by extended linkers. We find that Hv1 inhibition by the new compounds presents fast and slow binding modalities, which are affected by distinct types of perturbations of the binding site. The conservative amino acid substitution D112E in the Hv1 selectivity filter abolishes the fast component, leaving the slow component unaltered, whereas the F150A substitution affects primarily the slow component. Based on kinetic analysis of the inhibition process, we propose that the new arginine mimics are able to bind the open channel like 2GBI. However, unlike 2GBI, the new compounds become “trapped” inside the VSD when the gate closes, and they can slowly reach the binding site in the trapped conformation even from the closed state. These pharmacological properties are desirable features for the treatment of diseases associated with Hv1 gain of function.