Dooku1 (Yoda1 Analog)

Piezo1

在 HEK 293 和 CHO 细胞中，Dooku1（10 μM，300 秒）表现出对 Piezo1 通道的选择性 [1]。在 Piezo1 T-REx 细胞中，Dooku1（10 μM，140 秒）对 Piezo1 通道的组成型活性没有影响 [1]。在 HEK 293 和 Piezo1 T-REx 细胞中，Dooku1（10 μM，40-60 秒）会阻断内源性 Yoda1 激活通道 [1]。

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Yoda1吡嗪环的修饰产生了一种类似物，该类似物缺乏激动剂活性，但可逆地拮抗Yoda1。这种类似物被称为Dooku1。Dooku1抑制了2μM Yoda1诱导的Ca2+进入，IC50为1.3μM（HEK 293细胞）和1.5μM（HUVEC），但未能抑制组成型Piezo1通道活性。它对内源性ATP诱发的HEK 293细胞中Ca2+升高或储存操作的Ca2+进入，或通过CHO和HEK 293电池中过表达的TRPV4或TRPC4通道进入Ca2+没有影响[1]。

Dooku1（10 μM 动脉松弛 20 分钟）阻断可降低 Yoda1 诱导的野生型 C57BL/6 小鼠的主动脉松弛 [1]。

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Dooku1抑制Yoda1诱导的主动脉舒张[1]
为了确定Dooku1是否抑制Yoda1引起的舒张，将主动脉环与10μM Dooku1预孵育20分钟。Dooku1强烈抑制了Yoda1诱导的舒张（图8A-C）。为了更详细地描述这一现象，我们在主动脉测定中测试了另外四种Yoda1类似物。所选的类似物显示出抑制Piezo1-T-REx细胞中Yoda1反应的各种能力：类似物2e（无激活和无抑制）（图1）、2g（轻微激活和轻微抑制）（见图1）、7b（轻微活化和部分抑制）（附图2和3）和11。模拟2e没有影响（图8D-F）。相比之下，2g、7b和11抑制了Yoda1诱导的弛豫（图8G-K）。此外，这些类似物抑制Yoda1诱导的弛豫的能力与抑制Yoda 1诱导的Ca2+进入相关（图8L）。数据表明，Dooku1作为Yoda1诱导的主动脉舒张抑制剂具有很强的疗效，该舒张是通过破坏Yoda1诱发的Piezo1通道活性介导的。

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Dooku1对Yoda1诱导的舒张具有选择性，但部分抑制激动剂收缩反应[1]
在Dooku1存在的情况下对PE反应的分析显示，对基线张力没有影响，但有显著的抑制作用（图9A，B）。为了确定Dooku1对PE诱导的收缩的抑制是否是这种收缩剂特有的，我们还测试了Dooku1对抗Tx A2模拟物U46619诱导的收缩作用。主动脉环用0.1μM U46619预收缩（图9C，D）。添加Dooku1导致部分松弛（图9D，E）。相比之下，Dooku1对ACh（1μM）或no供体SIN-1（10μM）诱发的舒张没有影响（图9F，G）。在其他四种Yoda1类似物存在的情况下，对PE反应的研究表明没有抑制作用（图10）。数据表明，Dooku1选择性抑制Yoda1诱导的舒张，但也通过未知机制部分抑制受体介导的激动剂反应。

细胞内Ca2+测量[1]
实验前24小时，将HEK 293和CHO细胞以90%的融合率铺在聚赖氨酸涂层96孔板（美国纽约州康宁）中，将HUVEC铺在透明96孔板中。在标准浴溶液（SBS）中的0.01%普朗尼克酸存在下，将细胞与2μM fura-2-AM或4μM fluo-4-AM（用于表达TRPV4的CHO细胞）在37°C下孵育1小时。对于氟-4的记录，在整个实验过程中，SBS中包括2.5mM丙磺舒。在室温下用SBS洗涤细胞30分钟。如果正在测试抑制剂，则应在SBS洗涤后立即添加这些抑制剂，并在实验的其余部分进行维护。在室温下，在Softmax Pro软件v5.4.5控制的96孔荧光板阅读器上进行测量。对于使用fura-2的记录，细胞内钙的变化（Δ）表示为340和380 nm激发下fura-2发射（510 nm）强度的比值。对于使用fluo-4的记录，染料在485nm处被激发，在525nm处发出收集的光，测量结果以任意单位显示为绝对荧光。SBS含有（mM）：130 NaCl、5 KCl、8 D-葡萄糖、10 HEPES、1.2 MgCl2、1.5 CaCl2，用NaOH滴定pH至7.4。对于Ca2+加反实验，使用无Ca2+的SBS（不含CaCl2），Ca2+加回为0.3 mM。对于洗脱实验，在记录前立即用SBS洗涤抑制剂3次。

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FluxOR™细胞内Tl+（铊离子）测量[1]
实验前24小时，将诱导（Tet+）和非诱导（Tet-）Piezo1 HEK 293细胞以90%的融合率铺在聚赖氨酸涂层的96孔板上，将HUVEC铺在透明96孔板中。细胞在室温下用FluxOR染料加载1小时，然后转移到测定缓冲液中20分钟。如果正在测试抑制剂，则此时添加这些抑制剂并在整个实验过程中保持。根据制造商的说明，用含Tl+的无K+溶液刺激细胞。在室温下，在Softmax Pro软件v5.4.5控制的96孔荧光板阅读器上进行测量。FluxOR在485nm处激发，在520nm处收集发射光，测量值表示为比基线增加的比率（F/F0）。

细胞活力测定 [1]
细胞类型： HUVEC、Piezo1 T-REx 细胞
测试浓度： 10 μM
孵育时间： 40-60 s
实验结果： 对Yoda1诱导的Ca2+进入HUVEC具有浓度依赖性抑制作用，IC50为1.49 μM。在 HUVEC 中的效力增强，EC50 为 0.23 μM，在 Piezo1 T-REx 细胞中的效力增强，EC50 为 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

80

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

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In solvent   -80°C    6 months

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