Jaquet, V.; Penttinen, R.; Rodriguez, G.; Castelbou, C.; Cambet, Y.; Rosa, N.; Bourdin, M.; Asghariastanehei, B.; de Lima, F.; Martin, M.; Nader, E.; Serra, S.; Fernandez-Fernandez, J.; Murciano, N.; Connes, P.; Egee, S.; Guizouarn, H.; Kaestner, L.; Fertig, N.; Rotordam, M. G.; Demaurex, N.

Background and purpose PIEZO1 mechanosensitive cation channels translate mechanical cues into intracellular Ca2+; and Na+; elevations, enabling cells to respond to physical alterations in their environment. PIEZO1 contributes to red blood cells (RBC) volume homeostasis and gain-of-function PIEZO1 mutations cause hereditary xerocytosis (HX), a rare mostly compensated hemolytic anemia, and aberrant channel activation exacerbates sickling and vascular dysfunction in sickle cell disease. Despite strong genetic and physiological evidence supporting PIEZO1 as a therapeutic target, potent and selective inhibitors are limited, and existing compounds show modest specificity or poorly explored mechanisms. Improved pharmacological tools are needed. Experimental approach We conducted a high-throughput screen of FDA-approved drugs to identify PIEZO1 inhibitors. Compounds were tested at concentrations of 10 microM in a monocytic cell line, using intracellular Ca2+; elevations evoked by the PIEZO1 agonist Yoda1 as read-out. The inhibitory activity of the best hit was validated and compared to existing PIEZO1 inhibitors using electrophysiological analysis, orthogonal PIEZO1-dependent assays across cell lines and human RBCs. As functional proof, we investigated the impact of three PIEZO1 inhibitors on RBC deformability by ektacytometry, after Yoda1 pre-stimulation. Key results This screen identified Otenabant, a selective Cannabinoid Receptor Type 1 (CB1) antagonist, as a potent PIEZO1 inhibitor. Otenabant dose-dependently inhibited Ca2+ elevations mediated by endogenous or exogenously expressed human PIEZO1, but was ineffective against mouse Piezo1, revealing species-specific channel differences. Otenabant inhibited mechanosensitive currents elicited by shear stress in fibroblasts and by repeated poking in PIEZO1-expressing HEK-293 cells, altering the currents activation and inactivation kinetics, and prevented Yoda1-induced hyperpolarization in RBCs. Otenabant was able to reverse the negative impact of Yoda1 on RBC deformability. Conclusions and implications These findings demonstrate the utility of Yoda-based screening for discovering PIEZO1 antagonists and identify Otenabant as a promising chemical scaffold for developing selective PIEZO1 inhibitors with therapeutic potential.