ERK1/2

Akt and ERK1/2 are activated in a Piezo1-independent manner by Yoda1 (0–6 μM, 5 min) [2]. Rac1 activation is inhibited by Yoda1 (1.5 μM, 5 min) [3].

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Inhibition or activation of Piezo1 affects the apoptosis of HT22 cells in vitro ICH model [1]
The in vitro ICH model was established according to Wang et al. (2022). We first treated the in vitro cultured HT22 cells with a hemin solution of each of the different concentrations (0, 10, 20, 40, 60, 80, 100, 120, or 140 μM) to find out the IC50 of hemin. We found that the IC50 of hemin was 100 μM (Fig. 6a). We then used hemin solution at the IC50 (100 μM) to treat HT22 cells for 24 h to observe the effect of hemin on the viability of HT22 cells and protein levels of the indicated genes in HT22 cells. The abnormal morphological changes of HT22 cells that became smaller and rounder were ostensibly observed as early as 1 h after hemin treatment (Fig. 6b). Quantitative analysis of live cell counts showed a 50% reduction in the number of viable HT22 cells 24 h after hemin treatment (Fig. 6c). Yoda-1 (a Piezo1 activator) treatment further decreased the number of viable HT22 cells that were pre-treated with hemin. In contrast, GsMTx4 (Piezo1 blocker) treatment significantly reversed the massive reduction in the number of viable HT22 cells after hemin pre-treatment (Fig. 6c).

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Yoda-1 induces both Akt and ERK1/2 activation in endothelial cells. Activation of Akt, but not ERK1/2, by Yoda-1 is abrogated by gadolinium (Gd3+). Ruthenium red (RR) effectively blocks Yoda1-induced activation of Akt. GsMTx4, a potent blocker of Piezo1, does not inhibit Yoda1-induced Akt or ERK1/2 phosphorylation.[2]

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Inhibition of EGF-stimulated macropinocytosis by Yoda-1 is dependent on Piezo1. KCa3.1 activation is necessary for the inhibitory effect of Yoda-1 on ruffle formation. In this study, we showed that Yoda-1 treatment led to inhibition of Rac1 activation, which inhibited peripheral membrane ruffle formation (Fig. 3). It was reported that knockdown of Piezo1 in gastric cancer cells led to Rac1 activation51. This previous study may suggest that Piezo1 activation inhibits Rac1, although the mechanism still remains unclear. Importantly, we further showed that the inhibitory effect of Yoda1 on macropinocytosis was dependent on extracellular Ca2+ influx through Piezo1 (Fig. 4). The inhibition of KCa3.1, which is a calcium-activated potassium channel, recovered the EGF-stimulated membrane ruffle formation (i.e. actin rearrangement) even in the presence of Yoda1 (Fig. 5A,B). This suggests that EGF-stimulated actin rearrangement can be induced even in the presence of Yoda1 on condition that KCa3.1 is inhibited. Therefore, we propose that Piezo1 activation followed by KCa3.1 activation likely leads to the inhibition of actin rearrangement. A previous study reported that KCa3.1 activation is essential in membrane ruffle closure, the later stage of macropinocytosis process21. On the other hand, we showed that a KCa3.1 activator as well as Yoda1 also impaired macropinocytosis (Fig. 5C–E). Altogether, our results suggest that appropriate temporal activation of KCa3.1 is important in macropinocytosis, and that KCa3.1 activation, following acute Ca2+ influx induced by Yoda1, could lead to the inhibition of actin rearrangement (Fig. 5F) [3].

Secondary brain injury after intracerebral hemorrhage (ICH) is the main cause of poor prognosis in ICH patients, but the underlying mechanisms remain less known. The involvement of Piezo1 in brain injury after ICH was studied in a mouse model of ICH. ICH was established by injecting autologous arterial blood into the basal ganglia in mice. After vehicle, Piezo1 blocker, GsMTx4, Piezo1 activator, Yoda-1, or together with mannitol (tail vein injection) was injected into the left lateral ventricle of mouse brain, Piezo1 level and the roles of Piezo1 in neuronal injury, brain edema, and neurological dysfunctions after ICH were determined by the various indicated methods. Piezo1 protein level in neurons was significantly upregulated 24 h after ICH in vivo (human and mice). Piezo1 protein level was also dramatically upregulated in HT22 cells (a murine neuron cell line) cultured in vitro 24 h after hemin treatment as an in vitro ICH model. GsMTx4 treatment or together with mannitol significantly downregulated Piezo1 and AQP4 levels, markedly increased Bcl2 level, maintained more neurons alive, considerably restored brain blood flow, remarkably relieved brain edema, substantially decreased serum IL-6 level, and almost fully reversed the neurological dysfunctions at ICH 24 h group mice. In contrast, Yoda-1 treatment achieved the opposite effects. In conclusion, Piezo1 plays a crucial role in the pathogenesis of brain injury after ICH and may be a target for clinical treatment of ICH.[4]

384-well format [1]
2 days after transfection, the cells were washed with assay buffer (1× HBSS, 10 mM HEPES, pH7.4) using a ELx405 CW plate washer. Cells were incubated with assay buffer containing 4 μM Fluo3 and 0.04% Pluronic F-127 for ∼60 min and then washed again with assay buffer. Fluorescence was monitored on a fluorescent imaging plate reader (FLIPR) Tetra. To chelate extracellular calcium (1× HBSS contains 1.26 mM CaCl2), 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the cells 1 min before addition of the indicated Yoda1 concentration in presence of 2 mM EGTA. To deplete intracellular calcium, 7.5 μM thapsigargin was added 15 min before Yoda1 addition. A 10-mM stock solution of Yoda1 in dimethyl sulfoxide (DMSO) was used resulting in a maximum of 1% DMSO in the assay. Concentration-response curves were fitted using a sigmoidal dose–response at variable slope.[1]

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1536-well high-throughput screen format [1]
With objective of identifying either a Piezo1 or Piezo2 agonist, we co-transfected cells with mPiezo1 and mPiezo2 cDNA at equal amounts. 2 days after, transfection cells were incubated with Calcium5 according to manufacturer's instruction and fluorescence was monitored on FLIPR Tetra. About 3.25 million compounds from the LMW Novartis screening library, which includes public domain and proprietary drug-like molecules, were screened at a concentration of 5 μM. Approximately 9000 hits, as defined by 50% activation above DMSO control wells, were selected for retesting in co-transfected cells as well as individual Piezo1 and 2 transfection and control cells. From this, Yoda1 was identified as a potential Piezo1 activator and selected for further study.

Western Blot analysis
Cell Types: human coronary artery endothelial cells (HCAEC) [2], A431 cells [3]
Tested Concentrations: 0, 1.5, 3.0 and 6.0 μM
Incubation Duration: 5 minutes
Experimental Results: Induced Akt and ERK1/2 activation, and increased Phosphorylation levels of Akt and ERK1/2 were dose-dependent. Inhibits EGF-induced increase in Rac1-GTP amount and inhibits Rac1 activation.

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In vitro intracerebral hemorrhage (ICH) model [4]
The in vitro ICH model was simulated by hemin treatment of in vitro cultured HT22 cells (a murine hippocampal neuron cell line). According to Wang et al. (2022), the HT22 cells were pre-cultured with DMEM (10% fetal bovine serum and 1% Penicillin-Streptomycin) for 12 h. Then, a hemin solution with each of the different concentrations (0, 10, 20, 40, 60, 80, 100, 120, or 140 μM) was added to the cell culture medium with an additional 24 h culture of HT22 cells. For GsMTx4 or Yoda-1 treatment group, GsMTx4 or Yoda-1 was added 1 h after hemin addition to HT22 cells.

Inhibition or activation of Piezo1 after intracerebral hemorrhage (ICH) in mice[4]
The effects of Piezo1 blocker, GsMTx4, and Piezo1 activator, Yoda-1, on brain edema and neurological dysfunctions after ICH were studied in 27 mice. The mice were divided into five groups, (1) control (n = 6), (2) ICH 24 h (n = 6), (3) ICH 72 h (n = 3), (4) ICH 24 h + GsMTx4 (3 μM, n = 6), and (5) ICH 24 h + Yoda-1 (10 μM, n = 6). Please refer to the Supplementary Materials and Methods file (Appendix A Supplementary data) for details.

[1]. Chemical activation of the mechanotransduction channel Piezo1. Elife. 2015 May 22:4:e07369.

[2]. Yoda1-induced phosphorylation of Akt and ERK1/2 does not require Piezo1 activation. Biochem Biophys Res Commun. 2018 Feb 26;497(1):220-225.

[3]. Piezo1 activation using Yoda1 inhibits macropinocytosis in A431 human epidermoid carcinoma cells. Sci Rep. 2022 Apr 15;12(1):6322.

[4]. Roles of mechanosensitive ion channel PIEZO1 in the pathogenesis of brain injury after experimental intracerebral hemorrhage. Neuropharmacology . 2024 Jun 15:251:109896.

Yoda 1 is a member of the class of thiadiazoles that is 1,3,4-thiadiazole substituted by pyrazin-2-yl and (2,6-dichlorobenzyl)sulfanediyl groups at positions 2 and 5, respectively. It is a selective activator of mechanosensitive channel piezo1. It has a role as a glycine transporter 2 inhibitor and a piezo1 agonist. It is an aromatic compound, a member of pyrazines, a member of thiadiazoles, an organic sulfide and a dichlorobenzene.

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Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ~3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function. [1]

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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. [2]

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Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools. [3]

C13H8CL2N4S2

355.265417098999

353.956

C, 43.95; H, 2.27; Cl, 19.96; N, 15.77; S, 18.05

448947-81-7

448947-81-7;

2746822

White to light yellow solid powder

1.6±0.1 g/cm3

538.4±60.0 °C at 760 mmHg

279.4±32.9 °C

0.0±1.4 mmHg at 25°C

1.714

4.89

0

6

4

21

329

0

C1=CC(=C(C(=C1)Cl)CSC2=NN=C(S2)C3=NC=CN=C3)Cl

BQNXBSYSQXSXPT-UHFFFAOYSA-N

InChI=1S/C13H8Cl2N4S2/c14-9-2-1-3-10(15)8(9)7-20-13-19-18-12(21-13)11-6-16-4-5-17-11/h1-6H,7H2

2-[5-(2,6-Dichloro-benzylsulfanyl)-[1,3,4]thiadiazol-2-yl]-pyrazine

Yoda1; Yoda-1; GlyT2-IN-1; YODA-1; 2-((2,6-dichlorobenzyl)thio)-5-(pyrazin-2-yl)-1,3,4-thiadiazole; TW6GF9RW6S; 2-[(2,6-dichlorophenyl)methylsulfanyl]-5-pyrazin-2-yl-1,3,4-thiadiazole; Yoda 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~15.62 mg/mL (~43.97 mM)
Ethanol : ~5 mg/mL (~14.07 mM)

Solubility in Formulation 1: ≥ 1.56 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.56 mg/mL (4.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)