Yoda1

Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation

Background and purpose: 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. Experimental approach: 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. Key results: 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 IC50 s 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. Conclusion and implications: 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.

A mechanism for the activation of the mechanosensitive Piezo1 channel by the small molecule Yoda1

Mechanosensitive Piezo1 and Piezo2 channels transduce various forms of mechanical forces into cellular signals that play vital roles in many important biological processes in vertebrate organisms. Besides mechanical forces, Piezo1 is selectively activated by micromolar concentrations of the small molecule Yoda1 through an unknown mechanism. Here, using a combination of all-atom molecular dynamics simulations, calcium imaging and electrophysiology, we identify an allosteric Yoda1 binding pocket located in the putative mechanosensory domain, approximately 40 ? away from the central pore. Our simulations further indicate that the presence of the agonist correlates with increased tension-induced motions of the Yoda1-bound subunit. Our results suggest a model wherein Yoda1 acts as a molecular wedge, facilitating force-induced conformational changes, effectively lowering the channel's mechanical threshold for activation. The identification of an allosteric agonist binding site in Piezo1 channels will pave the way for the rational design of future Piezo modulators with clinical value.

Yoda1's energetic footprint on Piezo1 channels and its modulation by voltage and temperature

Piezo1 channels are essential mechanically activated ion channels in vertebrates. Their selective activation by the synthetic chemical activator Yoda1 opened new avenues to probe their gating mechanisms and develop novel pharmaceuticals. Yet, the nature and extent of Piezo1 functions modulated by this small molecule remain unclear. Here we close this gap by conducting a comprehensive biophysical investigation of the effects of Yoda1 on mouse Piezo1 in mammalian cells. Using calcium imaging, we first show that cysteine bridges known to inhibit mechanically evoked Piezo1 currents also inhibit activation by Yoda1, suggesting Yoda1 acts by energetically modulating mechanosensory domains. The presence of Yoda1 alters single-channel dwell times and macroscopic kinetics consistent with a dual and reciprocal energetic modulation of open and shut states. Critically, we further discovered that the electrophysiological effects of Yoda1 depend on membrane potential and temperature, two other Piezo1 modulators. This work illuminates a complex interplay between physical and chemical modulators of Piezo1 channels.

Tubeimoside I Antagonizes Yoda1-Evoked Piezo1 Channel Activation

Piezo1, a mechanosensitive Ca²⁺-permeable non-selective cationic ion channel protein, is involved in a wide range of biological processes and plays crucial roles in vascular development. However, the pharmacology of this protein is in its infancy. Yoda1, the first specific chemical activator of Piezo1 channels, can activate Piezo1 in absence of mechanical stimulation. Hence, we sought to identify inhibitors of Yoda1 from Traditional Chinese Medicine (TCM). Intracellular Ca²⁺ measurements were conducted in human umbilical vein endothelial cells (HUVECs), HEK 293T cells overexpressing TRPC5 and TRPM2 channels, as well as in CHO K1 cells overexpressing TRPV4 channels. We identified tubeimoside I (TBMS1) as a strong inhibitor of the Yoda1 response and demonstrated its selectivity for the Piezo1 channels. Similarly, Yoda1-induced inhibitory results were obtained in Piezo1 wild-type overexpressed cells, murine liver endothelial cells (MLECs), and macrophages. The physiological responses of TBMS1 were identified by isometric tension, which can inhibit Yoda1 relaxation of aortic rings. Our results demonstrated that TBMS1 can effectively antagonize Yoda1 induced Piezo1 channel activation. This study sheds light on the existence of Yoda1 inhibitors and improves the understanding of vascular pharmacology through Piezo1 channels.

Yoda1-induced phosphorylation of Akt and ERK1/2 does not require Piezo1 activation

Piezo1 is a mechanosensitive cation channel that is activated by shear stress in endothelial cells (ECs). It has been shown to mediate shear-induced EC responses, including increased calcium influx, and vascular functions, such as vascular tone and blood pressure. Yoda1, a selective Piezo1 activator, has been shown to mimic shear-induced responses in ECs. Since shear-induced calcium influx causes Akt and ERK1/2 activation in ECs, we examined the effects of Yoda1 and the role of Piezo1 on their activation. Here, we show that Yoda1 robustly activates Akt and ERK1/2 in ECs. Additionally, the Piezo1 antagonists, gadolinium and ruthenium red, but not GsMTx4, effectively blocks Yoda1-induced Akt activation. Our results suggest that Yoda1-induced Akt and ERK1/2 activation is not dependent on Piezo1.