BPS2026 – A high-throughput screening method identifies marine-derived small molecule inhibitors of Hv1 channels

Human voltage-gated proton channels (Hv1) play a critical role in regulating intracellular pH and thereby the release of reactive oxygen species (ROS) and proinflammatory cytokines from neutrophils and microglia. Hv1 is therefore an attractive therapeutic target for suppression of inflammatory diseases such as neutrophil-mediated acute lung injury (ALI) and microglial-mediated neuroinflammation and neurodegeneration. We have previously demonstrated suppression of ALI using the high-affinity and specific Hv1 inhibitor C6 peptide, but no similarly effective small molecules have been described. We established a high-throughput screening (HTS) platform incorporating a pH-sensitive fluorescent protein fused to Hv1, enabling efficient functional screening in a 96-well plate format. Using this system, we interrogated a library of marine natural products derived from algae, cyanobacteria, and sponges, together with synthetic analogs, to identify novel Hv1 inhibitors. From this screen, we discovered four previously uncharacterized Hv1 inhibitors. Among these, F6, a thiazolidine derivative demonstrated nanomolar potency (88 nM at 0 mV) that stabilizes the closed-channel conformation. Site-directed mutagenesis indicates the channel binding site as the S3-S4 loop, a conclusion supported by AlphaFold3 modeling. Preliminary structure-activity relationship studies delineate the pharmacophoric features of F6 essential for Hv1 inhibition. Confirming its activity in vitro, F6 attenuated both ROS production and proinflammatory cytokine release in induced pluripotent stem cell (iPSC)-derived microglia, underscoring its capacity to mitigate neuroinflammation. This study reports the first marine-derived small-molecule Hv1 inhibitors, establishes a robust HTS platform for Hv1-targeted drug discovery, and identifies F6 as a promising lead compound for the treatment of Hv1-associated inflammatory disorders.