BPS2026 – A platform to study the effects of small molecules on Hv1 function in neutrophils

The Hv1 voltage-gated proton channel plays an important role in infection and inflammation. For example, it was recently shown to regulate corneal recruitment of neutrophils in a mouse model of eye infection by Pseudomonas aeruginosa . Neutrophils utilize a variety of effectors to kill invading pathogens. Some of these effectors, such as proinflammatory cytokines, reactive oxygen species (ROS), and proteolytic enzymes, can also lead to serious collateral tissue damage that persists even post-infection. Antibiotic treatment alone is often insufficient to prevent the damage caused by neutrophils, hence the need for drugs that can minimize such collateral damage. The role of Hv1 in modulating neutrophils immune response makes it a promising molecular target for this type of drugs. Here, we developed synthetic strategies to generate the Hv1 inhibitor HIF and other new 2-aminoimidazole derivatives, and to prepare inactive isosteric compounds that can be used as negative controls in pharmacological studies targeting Hv1. We characterized the new derivatives through electrophysiological measurements performed on human Hv1 expressed in a heterologous system. We also employed a CRISPR-Cas9 approach to knockout the Hv1 gene (HVCN1) in HL-60 cells, a human promyelocytic leukemia cell line that can be differentiated into neutrophil-like cells in vitro. We used the differentiated cells to study the effects of HIF and its analogs on neutrophil function and to compare pharmacological Hv1 inhibition to genetic Hv1 ablation. We then validated the results using human neutrophils isolated from peripheral blood of healthy subjects.