The mechanosensitive Piezo1 channel has important roles in vascular physiology and disease. Yoda1 is a small‐molecule agonist, but the pharmacology of these channels is otherwise limited.

Yoda1 analogues were generated by synthetic chemistry. Intracellular Ca²⁺ and Tl⁺ measurements were made in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVECs, which natively express Piezo1. Isometric tension recordings were made from rings of mouse thoracic aorta.

Modification of the pyrazine ring of Yoda1 yielded an analogue, which lacked agonist activity but reversibly antagonized Yoda1. The analogue is referred to as Dooku1. Dooku1 inhibited 2 μM Yoda1‐induced Ca²⁺‐entry with IC₅₀s of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs) yet failed to inhibit constitutive Piezo1 channel activity. It had no effect on endogenous ATP‐evoked Ca²⁺ elevation or store‐operated Ca²⁺ entry in HEK 293 cells or Ca²⁺ entry through TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 caused dose‐dependent relaxation of aortic rings, which was mediated by an endothelium‐ and NO‐dependent mechanism and which was antagonized by Dooku1 and analogues of Dooku1.

Chemical antagonism of Yoda1‐evoked Piezo1 channel activity is possible, and the existence of a specific chemical interaction site is suggested with distinct binding and efficacy domains.