HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD–ligand interactions

Abstract

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.

A novel Hv1 inhibitor reveals a new mechanism of inhibition of a voltage-sensing domain

“…Mutagenesis was performed as previously described ( Hong et al, 2013 ). mRNAs were synthesized using mMESSAGE mMACHINE T7 Transcription Kit (Ambion) or HiScribe T7 ARCA mRNA Kit (with tailing; New England Biolabs) from linearized DNA constructs described in Zhao et al (2021) . All chemical reagents were at the highest purity commercially available.…”

“…ABI and 2GBI were from Sigma-Aldrich. HIF, 3-(2-amino-5-methyl-1H-imidazol-4-yl)-1-phenyl-propan-1-one (HIF NF ), N-[(2-amino-5-methyl-1H-imidazol-4-yl)methyl]-3,5-difluorobenzamide (HIF NH ), 3-(2-amino-5-methyl-1H-imidazol-4-yl)-1-(3,5-difluorophenyl)propan-1-ol (HIF OH ), and (2 E )-3-(2-amino-5-methyl-1H-imidazol-4-yl)-1-(3,5-difluorophenyl)prop-2-en-1-one (HIF EN ) were custom synthesized by Enamine (see Zhao et al, 2021 ). With the exception of HIF NH , all HIF compounds were in the form of hydrochloride salt.…”

“…The starting pose for MD simulations was chosen from visual evaluation of the ligand’s proximity to putative binding site residues based on experimental data (proximity of the five-membered ring to D174 and proximity of the fluorinated ring to W207). HIF was parameterized as described in Zhao et al (2021) . MD simulations of HIF-bound Hv1 VSD embedded in a solvated lipid bilayer made of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were performed using NAMD 2.13 ( Phillips et al, 2005 ).…”

“…Both the imidazole ring and the condensed phenyl ring of the compound were found to interact with the channel ( Hong et al, 2014 ). To improve such interactions, a new class of inhibitors was generated in which the two rings were separated by flexible linkers ( Zhao et al, 2021 ). These compounds, including 3-(2-amino-5-methyl-1H-imidazol-4-yl)-1-(3,5-difluorophenyl)propan-1-one (HIF), were also compared with 2-aminobenzimidazole (ABI) derivatives to assess the effect of fluorination on the ligand apparent binding affinity.…”

“…These compounds, including 3-(2-amino-5-methyl-1H-imidazol-4-yl)-1-(3,5-difluorophenyl)propan-1-one (HIF), were also compared with 2-aminobenzimidazole (ABI) derivatives to assess the effect of fluorination on the ligand apparent binding affinity. HIF, which contains a difluorophenyl ring, was found to be superior to both ABI and 2GBI at inhibiting Hv1, and its binding site within the VSD in the open conformation was proposed to overlap with the 2GBI binding site ( Zhao et al, 2021 ).…”