5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel

Abstract

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 μM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 μM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 μM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Identification of a Novel Structural Class of HV1 Inhibitors by Structure-Based Virtual Screening

“…The so-called “H V 1 inhibitor flexible” compounds (HIFs) (Figure B) were developed using structural modifications of guanidine derivatives to better explore the binding site for potential stabilizing interactions while reducing the overall hydrophobicity. Some of the HIF compounds have been reported to exhibit stronger inhibitory properties than guanidinobenzimidazoles. , However, the selectivity of these compounds over other voltage-gated ion channels is low and their limited ability to penetrate the cell membrane makes them unsuitable for in vivo experiments. ,, Recently, using a structure-based approach, YHV98–4 (Figure C) was identified as a selective voltage-gated proton channel inhibitor that binds to an intermediate conformational state of the protein, a transition state between the resting and activated states . By blocking H V 1-mediated currents in DRG neurons, YHV98–4 has been shown to reduce chronic pain and, at micromolar concentrations, has an anti-inflammatory effect that may reduce morphine-induced adverse effects .…”

“… 32 , 33 However, the selectivity of these compounds over other voltage-gated ion channels is low and their limited ability to penetrate the cell membrane makes them unsuitable for in vivo experiments. 1 , 20 , 34 Recently, using a structure-based approach, YHV98–4 ( Figure 1 C) was identified as a selective voltage-gated proton channel inhibitor that binds to an intermediate conformational state of the protein, a transition state between the resting and activated states. 5 By blocking H V 1-mediated currents in DRG neurons, YHV98–4 has been shown to reduce chronic pain and, at micromolar concentrations, has an anti-inflammatory effect that may reduce morphine-induced adverse effects.…”