Piezo1

In HEK 293 and CHO cells, Dooku1 (10 μM, 300 seconds) exhibits selectivity for Piezo1 channels [1]. In Piezo1 T-REx cells, Dooku1 (10 μM, 140 seconds) had no influence on constitutive Piezo1 channel activity [1]. In HEK 293 and Piezo1 T-REx cells, Dooku1 (10 μM, 40–60 seconds) blocks endogenous Yoda1-activated channels [1].

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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[1].

Dooku1 (10 μM arterial relaxation for 20 min) blockade decreases Yoda1-induced aortic relaxation in wild-type C57BL/6 mice [1].

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Dooku1 inhibits Yoda1‐induced relaxation of aorta [1]
To determine if Dooku1 inhibits relaxation caused by Yoda1, aortic rings were pre‐incubated with 10 μM Dooku1 for 20 min. Dooku1 strongly suppressed the Yoda1‐induced relaxation (Figure 8A–C). To characterize this phenomenon in more detail, we tested four further Yoda1 analogues in the aorta assay. The selected analogues showed various abilities to inhibit Yoda1 responses in Piezo1 T‐REx cells: analogues 2e (no activation and no inhibition) (Figure 1), 2g (slight activation and slight inhibition) (Figure 1), 7b (slight activation and partial inhibition) (Figures 2 and 3) and 11 (slight activation and partial inhibition) (Figures 2 and 3). Analogue 2e had no effect (Figure 8D–F). 2g, 7b and 11 in contrast suppressed the Yoda1‐induced relaxation (Figure 8G–K). Moreover, the ability of these analogues to inhibit Yoda1‐induced relaxation correlated with inhibition of Yoda1‐induced Ca2+ entry (Figure 8L). The data suggest strong efficacy of Dooku1 as an inhibitor of Yoda1‐induced aortic relaxation that is mediated through disruption of Yoda1‐induced Piezo1 channel activity.

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Dooku1 is selective for Yoda1‐induced relaxation but partially inhibits agonist contractile responses [1]
Analysis of the PE response in the presence of Dooku1 revealed significant inhibition without effect on baseline tension (Figure 9A, B). To determine whether Dooku1's inhibition of PE‐induced contraction was specific to this contractile agent, we also tested the effect of Dooku1 against contraction induced by U46619, a Tx A2 mimetic. Aortic rings were pre‐contracted with 0.1 μM U46619 (Figure 9C, D). Addition of Dooku1 caused partial relaxation (Figure 9D, E). In contrast, Dooku1 had no effect on relaxation evoked by ACh (1 μM) or the NO donor SIN‐1 (10 μM) (Figure 9F, G). Investigation of the PE response in the presence of the other four Yoda1 analogues revealed no inhibitory effect (Figure 10). The data suggest that Dooku1 selectively inhibits Yoda1‐induced relaxation but also partially inhibits receptor‐mediated agonist responses via unknown mechanisms.

Intracellular Ca2+ measurements [1]
HEK 293 and CHO cells were plated in poly‐d‐lysine coated 96‐well plates (Corning, NY, USA) and HUVECs in clear 96‐well plates at a confluence of 90%, 24 h before experimentation. Cells were incubated with 2 μM fura‐2‐AM or 4 μM fluo‐4‐AM (for TRPV4 expressing CHO cells), in the presence of 0.01% pluronic acid in standard bath solution (SBS) for 1 h at 37°C. For recordings with fluo‐4, 2.5 mM probenecid was included in the SBS throughout the experiment. Cells were washed with SBS for 30 min at room temperature. If inhibitors were being tested, these were added at this time, immediately following an SBS wash and maintained during the rest of the experiment. Measurements were made at room temperature on a 96‐well fluorescence plate reader controlled by Softmax Pro software v5.4.5. For recordings using fura‐2, the change (Δ) in intracellular calcium was indicated as the ratio of fura‐2 emission (510 nm) intensities for 340 and 380 nm excitation. For recordings using fluo‐4, the dye was excited at 485 nm and emitted light collected at 525 nm, and measurements are shown as absolute fluorescence in arbitrary units. The SBS contained (mM): 130 NaCl, 5 KCl, 8 D‐glucose, 10 HEPES, 1.2 MgCl2, 1.5 CaCl2 and the pH was titrated to 7.4 with NaOH. For the Ca2+ add‐back experiments, Ca2+ free SBS was used (without CaCl2), and Ca2+ add‐back was 0.3 mM. For the washout experiments, inhibitors were washed 3 times with SBS immediately prior to recording.

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FluxOR™ intracellular Tl+ (thallium ion) measurements[1]
Induced (Tet+) and non‐induced (Tet−) Piezo1 HEK 293 cells were plated in poly‐d‐lysine coated 96‐well plates and HUVECs in clear 96‐well plates at a confluence of 90%, 24 h before experimentation. Cells were loaded with FluxOR dye for 1 h at room temperature, before being transferred to assay buffer for 20 min. If inhibitors were being tested, these were added at this time and maintained throughout the experiment. Cells were stimulated with a Tl+‐containing K+‐free solution according to the manufacturer's instructions. Measurements were made at room temperature on a 96‐well fluorescence plate reader controlled by Softmax Pro software v5.4.5. FluxOR was excited at 485 nm, emitted light collected at 520 nm, and measurements were expressed as a ratio increase over baseline (F/F0).

Cell viability assay [1]
Cell Types: HUVECs, Piezo1 T-REx Cell
Tested Concentrations: 10 μM
Incubation Duration: 40-60 s
Experimental Results: It has a concentration-dependent inhibitory effect on Yoda1-induced Ca2+ entry into HUVEC, with an IC50 of 1.49 μM. Potency was enhanced in HUVEC with an EC50 of 0.23 μM and in Piezo1 T-REx cells with an EC50 of 2.51 μM.

Animal/Disease Models: Wild-type male C57BL/6 mouse aortic ring [1]
Doses: 10 μM
Route of Administration: 20 minutes
Experimental Results: Inhibition of Yoda1-induced relaxation.

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Animals [1]
Twelve to sixteen week‐old, wild‐type male C57BL/6 mice were used for experiments. All mice were housed in GM500 individually ventilated cages at 21°C, 50–70% humidity and with a 12 h alternating light/dark cycle. They had ad libitum access to RM1 diet with bedding from Pure'o Cell. All animal experiments were authorized by the University of Leeds Animal Ethics Committee and the UK Home Office. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015).

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Aorta contraction studies[1]
The wire myograph technique using vessels from mice is regarded as a useful model for studying vascular reactivity (Outzen et al., 2015). Thoracic aorta was dissected out and immediately placed into ice‐cold Krebs solution (125 mM NaCl, 3.8 mM KCl, 1.2 mM CaCl2, 25 mM NaHCO3, 1.2 mM KH2PO4, 1.5 mM MgSO4, 0.02 mM EDTA and 8 mM D‐glucose, pH 7.4). Connective tissue and fat were carefully removed under a dissection microscope. Segments, 1 mm long, were mounted in an isometric wire myograph system with two 40 μm diameter stainless steel wires, bathed in Krebs solution at 37°C and bubbled with 95% O2, 5% CO2. The segment was then stretched stepwise to its optimum resting tension to a 90% equivalent transmural pressure of 100 mmHg and equilibrated for 1 h prior to experiments. The stretch was approximately equal to that expected at diastolic BP (Rode et al., 2017).

[1]. Yoda1 analogue (Dooku1) which antagonizes Yoda1-evoked activation of Piezo1 and aortic relaxation. Br J Pharmacol. 2018 May;175(10):1744-1759.

Background and Objectives: Mechanosensitive Piezo1 channels play an important role in vascular physiology and disease. Yoda1 is a small molecule agonist, but pharmacological studies of these channels are limited. Methods: A Yoda1 analogue was prepared using synthetic chemistry. Intracellular Ca2+ and Tl+ concentrations were measured in HEK 293 or CHO cell lines overexpressing channel subunits and in HUVEC cells naturally expressing Piezo1. Isometric tension was recorded from mouse thoracic aortic rings. Main Results: A Yoda1 analogue was obtained by modifying the pyrazine ring of Yoda1. This analogue lacked agonist activity but reversibly antagonized Yoda1. This analogue was named Dooku1. Dooku1 inhibited 2 μM Yoda1-induced Ca2+ influx with IC50 values of 1.3 μM (HEK 293 cells) and 1.5 μM (HUVECs cells), but failed to inhibit constitutive Piezo1 channel activity. It has no effect on endogenous ATP-induced Ca2+ elevation or storage-operated Ca2+ influx in HEK 293 cells, nor on Ca2+ influx mediated by TRPV4 or TRPC4 channels overexpressed in CHO and HEK 293 cells. Yoda1 induces dose-dependent relaxation of the aortic ring, an effect mediated by endothelium-dependent and NO-dependent mechanisms, and can be antagonized by Dooku1 and its analogues. Conclusion and significance: Yoda1-induced Piezo1 channel activity may be chemically antagonistic, suggesting the existence of specific chemical interaction sites with different binding and potency domains. [1]

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Currently, the only Piezo1 activity inhibitors are non-selective for Piezo1 (Drew et al., 2002; Bae et al., 2011). Dooku1 is not perfect either, as it does not directly block the channel, but it is a novel tool compound that can be used for Piezo1 characterization studies. It antagonizes the effects of Yoda1 and may contribute to understanding important small molecule binding sites on or near Piezo1 channels. Dooku1, without agonist activity, effectively inhibits Yoda1-induced Piezo1 activity. This inhibition of Piezo1 activity does not interfere with other intracellular Ca2+ processing processes or affect the effects of other aortic diastolic agents. Although these data suggest that Dooku1 is specific for Piezo1 channels, further research is needed to confirm this, especially considering that Dooku1 inhibits PE and U46619-induced aortic ring contraction, which may reflect a Piezo1 mechanism or other unknown roles of Dooku1. Dooku1 may act on Piezo1 in vascular smooth muscle cells, partially inhibiting contraction. This hypothesizes that these channels are activated during contraction through a Yoda1-like mechanism. Studies have found that Piezo1 is not essential for maintaining normal myogenic tone (Retailleau et al., 2015), therefore, the existence of non-Piezo1 targets for Dooku1 should be considered. [1]
Dooku1 is effective only for Yoda1-induced Piezo1 channel activity, but not for constitutive Piezo1 channel activity. This effect is consistent with the fact that Dooku1 acts on the same or similar sites as Yoda1, thereby blocking the binding of Yoda1 to its agonist binding sites. The reversibility of Dooku1 is consistent with that of Yoda1 (Rocio Servin-Vences et al., 2017). It would be beneficial to study whether the effect of Dooku1 conforms to competitive antagonism, but due to the solubility limitations of the compound, it is not possible to construct a suitable concentration-effect curve. Dooku1 has no effect on constitutive activity, indicating that the mechanism of its background channel activity is different from the mechanism of Yoda1 chemical activation. [1]
Dooku1 partially inhibits Yoda1 in HUVEC cells but strongly inhibits Yoda1 in the aorta (Fig. 6D vs. Fig. 8C). We initially speculated that this difference was due to the higher temperature of the contraction experiment (37°C vs. room temperature), but the temperature dependence of the Dooku1 effect was not significant (Fig. 3K). Another explanation may be that Ca2+ influx and NO production are not directly proportional, so partially inhibiting Yoda-1-induced Ca2+ influx is sufficient to inhibit most of the Yoda1-induced relaxation. Another difference is that Yoda1 is more potent in HUVEC cells than in Piezo1 T-REx cells, suggesting a difference between native Piezo1 channels and overexpressing Piezo1 channels (Fig. 6E, F). We speculate that this difference reflects a higher baseline activity state of channels in endothelial cells, as previously mentioned (Rode et al., 2017), making channels more sensitive to Yoda1 because they are more easily activated. [1] In summary, this study provides important insights into the structure-activity relationship of Yoda1 and supports the idea that there are specific chemical binding sites on or near the Piezo1 channel. In addition, this study also discovered a useful tool compound, Dooku1, which effectively antagonizes Yoda1-induced Piezo1 channel activity, thus distinguishing it from constitutive Piezo1 channel activity. The full role of Piezo1 in vascular biology is still under investigation, but the protein may have important clinical significance, playing an important role in genetic diseases, blood pressure control, hypertension-induced arterial remodeling, and exercise capacity (Retailleau et al., 2015; Wang et al., 2016; Rode et al., 2017). It is currently unclear whether activation or inhibition of the channel is more beneficial, but if the therapeutic potential of this protein is to be fully realized in the future, our understanding of the pharmacology and physiology of Piezo1 must be deepened. A deeper understanding of the interaction between Piezo1 channels and small molecules is expected to be an important aspect of a comprehensive understanding of Piezo1 biology. [1]

C13H9CL2N3OS

326.201059103012

324.984

2253744-54-4

137321150

White to off-white solid powder

3.8

1

4

4

20

316

0

ClC1C=CC=C(C=1CSC1=NN=C(C2=CC=CN2)O1)Cl

MNPOBXLPCWFONX-UHFFFAOYSA-N

InChI=1S/C13H9Cl2N3OS/c14-9-3-1-4-10(15)8(9)7-20-13-18-17-12(19-13)11-5-2-6-16-11/h1-6,16H,7H2

2-[(2,6-dichlorophenyl)methylsulfanyl]-5-(1H-pyrrol-2-yl)-1,3,4-oxadiazole

Dooku1; Dooku-1; Dooku 1; 2253744-54-4; 2-((2,6-Dichlorobenzyl)thio)-5-(1H-pyrrol-2-yl)-1,3,4-oxadiazole; 2-[(2,6-Dichlorobenzyl)thio)-5-(1H-pyrrol-2-yl)-1,3,4-oxadiazole; 2-{[(2,6-dichlorophenyl)methyl]sulfanyl}-5-(1H-pyrrol-2-yl)-1,3,4-oxadiazole; 2-((2,6-Dichlorobenzyl)thio)-5-(1H-pyrrol-2-yl)-1,3,4- oxadiazole; Dooku 1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~100 mg/mL (~306.56 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.38 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 20.8 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.)