Novel small-molecule modulation of PIEZO1 investigated by conventional and automated patch-clamp

PIEZO1 trimers form mechanically-activated calcium-permeable non-selective cation channels which are expressed in many types of cells. Mutations in the PIEZO1 gene are associated with generalised lymphatic dysplasia, varicose vein disease, dehydrated hereditary stomatocytosis, malarial resistance and other conditions. Pharmacological modulation of PIEZO1 has been achieved using various compounds, most notably the small-molecule agonist Yoda1 and its inhibitor Dooku1. However, improvements in the potency and pharmacokinetic properties of such compounds are required to increase the usefulness, and small-molecule inhibitors of PIEZO1 are largely lacking. We sought to identify novel small-molecule agonists and inhibitors of mouse and human PIEZO1 channels. Analogues of Yoda1 were generated by medicinal chemistry approaches and a library of 11,200 compounds was screened in a calcium assay using cells overexpressing human PIEZO1. Analogues of hits from the screen were designed and synthesised and then tested for effectiveness in cells overexpressing mouse or human PIEZO1 using an orthogonal calcium assay, conventional (manual) patch-clamp and automated patch-clamp in 384-well format (SyncroPatch 384). We identified Yoda1 analogues with better aqueous solubility and microsomal stability than Yoda1 and improved potency. We identified and validated two novel structurally distinct small-molecule inhibitors of PIEZO1. Automated patch-clamp techniques were refined for activation of PIEZO1 channels by fluid flow in 384-well format. Novel small-molecule modulators of the channels were tested in these automated assays and confirmed as PIEZO1 modulators. We gained preliminary knowledge of the structure-activity relationships and promising compounds for further development. Supported by British Heart Foundation.