Chemical modulation of the PIEZO1 mechanically-activated channel

Background PIEZO1 mechanically-activated Ca2+-permeable non-selective cation channels are expressed in a variety of eukaryotic cells. They have crucial roles in vascular and lymphatic development, red blood cell volume regulation, blood pressure regulation and physical performance. Mutations in the human PIEZO1 gene are implicated in anaemia, lymphatic disease and venous disease. PIEZO1 activation is achieved under mechanical burden such as shear stress, membrane stretching and membrane indentation. Pharmacological modulation has been achieved using entities including a spider toxin, ions, natural products, lipids and small-molecules, most notably the agonist Yoda1, and its successor Yoda2, and the Yoda1 derived inhibitor, Dooku1. Although these modulators have been valuable, barriers to progress have been the relative paucity and quality of inhibitors and weaknesses in technical capabilities for testing their effects. Aim This study sought to identify, characterise and develop small-molecule inhibitors of PIEZO1 channels and establish methodology to enable the routine testing of such molecules against mechanically activated PIEZO1 channel activity. Experimental Approach/Methods Small-molecules were designed and synthesised after initial screening. Their effects were tested against mouse and human PIEZO1 channels expressed in cells studied in a 96-well Ca2+i assay and by single cell and excised membrane patch-clamp. Automated patch-clamp methods were developed for routine testing of effects of molecules against mechanically-activated channel activity. Key Results Four novel structurally unique series of PIEZO1 inhibitors were identified. Analogue development and testing revealed preliminary structure activity relationships. Sub-threshold mechanical priming of PIEZO1 channels was discovered using new automated patch-clamp technology. Analogues with improved potency were identified using automated and manual patch-clamp and Ca2+ imaging techniques. Low micromolar inhibitors of the PIEZO1 channel were identified. Conclusions More effective small-molecule inhibition of PIEZO1 channels is now possible. Technology now exists for the routine investigation of inhibitor effects. PIEZO1 channels are susceptible to mechanical priming.