Otenabant is a Selective Antagonist of Human PIEZO1
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Background and purpose
PIEZO1 mechanosensitive cation channels translate mechanical cues into intracellular Ca 2+ 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 µM in a monocytic cell line, using intracellular Ca 2+ 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 Ca 2+ 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.
