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 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. [1]

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Currently, the only available inhibitors of Piezo1 activity are not selective for Piezo1 (Drew et al., 2002; Bae et al., 2011). Dooku1 is also not perfect as it does not directly block the channels, but it is a new tool compound that is useful for Piezo1 characterization studies. It antagonizes the action of Yoda1 and could facilitate understanding of an important small‐molecule binding site on or near to Piezo1 channels. Without agonist activity, Dooku1 effectively inhibits Yoda1‐induced Piezo1 activity. It does so without disturbing several Ca2+ handling events in the cell or affecting other aortic relaxing agents. Although these data suggest specificity of Dooku1 for Piezo1 channels, further studies to address this point are warranted, especially given the inhibitory effect of Dooku1 against PE and U46619‐induced contractions of aortic rings that might reflect a Piezo1 mechanism or some other unknown effect of Dooku1. It is possible that Dooku1 may be acting on Piezo1 in smooth muscle cells of the vessel, partially inhibiting contraction. This assumes that the channels become activated via a Yoda1‐like mechanism during contraction. Piezo1 was found not be required for normal myogenic tone (Retailleau et al., 2015), and so, a non‐Piezo1 target of Dooku1 should be considered. [1]

Dooku1 only has activity against Yoda1‐induced and not constitutive Piezo1 channel activity. Such an effect is consistent with Dooku1 acting at the same or a similar site to Yoda1 and thereby occluding access of Yoda1 to its agonist binding site. The reversibility of Dooku1 is consistent with the reversibility of Yoda1 (Rocio Servin‐Vences et al., 2017). It would be good to investigate if the Dooku1 effect is consistent with competitive antagonism, but solubility limitations of the compounds prevented construction of appropriate concentration–response curves. The inability of Dooku1 to have any effect on constitutive activity suggests that the mechanism of background channel activity is different to that of chemical activation with Yoda1.[1]

Dooku1 partially inhibited Yoda1 in HUVECs but strongly inhibited it in aorta (Figure 6D cf. Figure 8C). We initially speculated that the difference was due to the higher temperature of the contraction studies (37°C cf. room temperature), but the Dooku1 effect was not significantly temperature dependent (Figure 3K). An alternative explanation might be that Ca2+ entry is not directly proportional to NO production, so that partial inhibition of Yoda‐1 induced Ca2+ entry is sufficient to inhibit most of the relaxation induced by Yoda1. Another divergence was that Yoda1 was more potent in HUVECs than Piezo1 T‐REx cells, showing a difference between native and over‐expressed Piezo1 channels (Figure 6E, F). We speculate that this difference reflected a higher basal state of activity of the channels in endothelial cells, as described previously (Rode et al., 2017), making the channels more sensitive to Yoda1 because they are better primed for opening.[1]

In summary, this study has provided important insight into the structure–activity relationships of Yoda1 and supported the concept of a specific chemical binding site on or in close proximity to Piezo1 channels. It has also revealed the discovery of a useful tool compound, Dooku1, which effectively antagonizes Yoda1‐induced Piezo1 channel activity, distinguishing it from constitutive Piezo1 channel activity. The complete role of Piezo1 in vascular biology is still being established, but the protein may have significant clinical interest with emerging roles in genetic disease, BP control, hypertension‐induced arterial remodelling and exercise capacity (Retailleau et al., 2015; Wang et al., 2016; Rode et al., 2017). As yet, it is not clear whether activating or inhibiting this channel may be advantageous, but increasing our pharmacological knowledge, alongside our physiological knowledge of Piezo1 will be essential if therapeutic potential of this protein is to be harnessed in the future. Learning more about Piezo1 channel interactions with small‐molecules promises to be an important aspect of the overall effort to understand 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.)