Identification of a new family of inhibitors of the human Hv1 proton channel

The human voltage-gated proton channel (hHv1) plays an important role in immune and cancer cells being involved in functions such as proliferation, migration, and oxidative burst. The structure of this protein differs from that of other voltage-gated ion channels in that it has no conventional ion-conducting pore, conduction occurs through the voltage-sensing domain itself. This dissemblance may be the reason for the lack of selective hHv1 inhibitors. Our aim was to find potent and selective inhibitors for hHv1, which could be useful research tools and serve as lead molecules for the development of drug molecules targeting Hv1 for therapeutic goals. We used manual patch-clamp to test the Hv1-affinity and selectivity of potential inhibitors on CHO (Chinese hamster ovary) and HEK (human embryonic kidney) cells expressing hHv1 and other channels. Proliferation assays were performed to test possible off-target effects of the molecules. Over 30 small molecules were tested, of which seven turned out to be effective on hHv1 (i.e. blocked more than 50% of the current in 50 µM). Six of them had the same scaffold in their structure. The scaffold itself, named NZ-13, was also one of the hit molecules, and had an IC50 value of 26 µM for hHv1. Most of the hit molecules turned out to have low selectivity: they had comparable or even higher affinity for the other tested Kv and NaV ion channels than for Hv1. However, NZ-13 had lower affinity for the other channels than for Hv1 and also proved to have the smallest effect on T-cell proliferation. Based on our results, we have successfully identified a scaffold, which can be a starting point for creating new, potent and selective hHv1 inhibitors.