CAY10594
目录号 : GC18691
CAY10594 是一种有效的磷脂酶 D2 (PLD2) 抑制剂（体外 IC50 为 140 nm，细胞内为 110 nm）。
Cas No.:1130067-34-3
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
Human RPMI 8226 multiple myeloma B cells and human A431 skin epithelial carcinoma cells |
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Preparation Method |
Human RPMI 8226 multiple myeloma B cells and human A431 skin epithelial carcinoma cells were maintained in complete RPMI-1640 medium at 37 °C and 95 % air/5 % CO2. HEK-293 cells were maintained in complete DMEM/F12 medium. Cells in NaCl medium were pre-incubated at 37 °C for 15 min in the presence of DMSO, or 10 µM CAY10593, CAY10594 or halopemide. |
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Reaction Conditions |
10 µM for 500 seconds |
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Applications |
The PLD1 antagonist CAY10593 and to a lesser extent the PLD2 antagonist CAY10594 (both at 10 µM) significantly impaired ATP-induced CD23 shedding. The non-selective PLD antagonist and structural analogue of CAY10593, halopemide (10 µM) however had no significant effect on ATP-induced CD23 shedding. In the absence of ATP, no antagonist significantly altered cell-surface expression of CD23 compared to control-treated cells. |
| Animal experiment [2]: | |
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Animal models |
C57BL/6 mice |
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Preparation Method |
Mice were fasted for 16 hours before APAP injection. APAP (500 mg/kg) was administered with oral gavage in mice. CAY10594 was dissolved in 1% DMSO and intraperitoneally administered to mice 30 minutes prior to APAP injection for examining protective effects or after 3 hours from APAP challenge for investigating therapeutic effects of CAY10594. |
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Dosage form |
4/8/mg/kg |
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Applications |
Injection of APAP (500 mg/kg) caused marked liver injury, which was measured by hematoxylin and eosin staining of the livers. APAP-induced liver injury was almost completely blocked by the administration of a CAY10594 in a dose-dependent manner. APAP-induced hepatocyte death was measured by the TUNEL assay. Hepatocyte apoptosis was induced by APAP, which was also markedly decreased in CAY10594-administered mice compared with vehicle-treated mice. The protective effects of the CAY10594 against hepatocyte apoptosis were strongly induced at 4 or 8 mg/kg. |
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References: [1]. Pupovac A, Stokes L, Sluyter R. CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation[J]. Purinergic Signalling, 2013, 9(4): 609-619. | |
CAY10594 is an effective inhibitor of phospholipase D2 (PLD2) (IC50 140 nm in vitro and 110 nm in cells). Cay10594 highly inhibits the invasive migration of breast cancer cells in vitro and regulates the phosphorylation of GSK-3 β/ JNK axis ameliorates paracetamol induced acute liver injury.
CAY10594 significantly improved intestinal mucosal inflammation, which was characterized by higher survival rate, slight decrease in body weight, less or no bloody stool and lower pathological score level,compared with the control group. RNA was extracted from colon tissue to detect the expression of cytokines, as well as proinflammatory cytokines, such as TNF- α?￠ IL-6, IL-23 and IL-1 β It was found that PLD2 was significantly decreased after blocking in DSS induced colitis, while anti-inflammatory cytokines were significantly increased after inhibiting PLD2. In addition, fresh colon samples were also obtained and cultured in vitro for 24 hours; The supernatant was collected to detect cytokines by ELISA. Proinflammatory cytokines (e.g., IL-17A, TNF- α and IL-1 β) [1] While anti-inflammatory cytokines (for example, IL-10 was found to increase after PLD2 blockade), indicating that PLD2 blockade can improve intestinal mucosal inflammation [2].
CAY10594 induced a strong therapeutic effect in APAP challenged mice. The results show that PLD2 plays an important role in mediating APAP induced liver injury [3]. Because PLD2 has basic activity, the administration of cay10594 will block the production of PLD2 enzyme active product PA in an experimental acute liver injury model. Therefore, it is reasonable to assume that the protective and therapeutic effects of cay10594 in the acute liver injury model will be mediated by blocking the production of PA. Cay10594 may regulate early liver pathology to prevent APAP induced liver injury by rapidly restoring GSH levels without affecting antioxidant gene expression [4].
References:
[1] Lee S K, Kim S D, Kook M, et al. Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2[J]. Journal of Experimental Medicine, 2015, 212(9): 1381-1390.
[2] Zhou G, Yu L, Yang W, et al. Blockade of PLD2 ameliorates intestinal mucosal inflammation of inflammatory bowel disease[J]. Mediators of inflammation, 2016, 2016.
[3] Mitchell J R, Jollow D J, Potter W Z, et al. Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism[J]. Journal of Pharmacology and Experimental Therapeutics, 1973, 187(1): 185-194.
[4] Lee S K, Bae G H, Kim Y S, et al. A phospholipase D2 inhibitor, CAY10594, ameliorates acetaminophen-induced acute liver injury by regulating the phosphorylated-GSK-3β/JNK axis[J]. Scientific Reports, 2019, 9(1): 1-10.
CAY10594 是一种有效的磷脂酶 D2 (PLD2) 抑制剂（体外 IC50 为 140 nm，细胞内为 110 nm）。 Cay10594在体外高度抑制乳腺癌细胞的侵袭性迁移并调节GSK-3的磷酸化 β/JNK轴改善对乙酰氨基酚诱导的急性肝损伤。
CAY10594显着改善肠粘膜炎症，其特征是与对照组相比,存活率较高,体重轻度下降,血便较少或无,病理评分水平较低。从结肠组织中提取RNA，检测细胞因子的表达，以及促炎细胞因子，如TNF- α；. IL-6、IL-23和IL-1 β；发现在DSS诱导的结肠炎中阻断后PLD2显着降低，而抑制PLD2后抗炎细胞因子显着增加。此外，还取新鲜结肠标本，体外培养24小时；收集上清液以通过ELISA检测细胞因子。促炎细胞因子（例如，IL-17A、TNF-α；和 IL-1 β；）[1] 而抗炎细胞因子（例如，IL-10 在 PLD2 阻断后被发现增加） , 表明 PLD2 阻断可以改善肠粘膜炎症 [2]。
CAY10594 在 APAP 攻击的小鼠中诱导了强烈的治疗效果。结果表明PLD2在介导APAP诱导的肝损伤中起重要作用[3]。因为 PLD2 具有基本活性，cay10594 的给药将阻断实验性急性肝损伤模型中 PLD2 酶活性产物 PA 的产生。因此，可以合理地假设 cay10594 在急性肝损伤模型中的保护和治疗作用将通过阻断 PA 的产生来介导。 Cay10594 可能通过快速恢复 GSH 水平而不影响抗氧化基因表达来调节早期肝脏病理，从而预防 APAP 诱导的肝损伤[4]。
| Cas No. | 1130067-34-3 | SDF | |
| 化学名 | N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide | ||
| Canonical SMILES | O=C(NCCN1CCC2(C(NCN2C3=CC=CC=C3)=O)CC1)C4=CC(C=CC=C5)=C5C=C4 | ||
| 分子式 | C26H28N4O2 | 分子量 | 428.5 |
| 溶解度 | DMF: 20 mg/ml,DMSO: 20 mg/ml,DMSO:PBS(pH7.2) (1:1): 0.5 mg/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.3337 mL | 11.6686 mL | 23.3372 mL |
| 5 mM | 0.4667 mL | 2.3337 mL | 4.6674 mL |
| 10 mM | 0.2334 mL | 1.1669 mL | 2.3337 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
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A phospholipase D2 inhibitor, CAY10594, ameliorates acetaminophen-induced acute liver injury by regulating the phosphorylated-GSK-3β/JNK axis
We examined the role of phospholipase D2 (PLD2) on acetaminophen (APAP)-induced acute liver injury using a PLD2 inhibitor (CAY10594). 500 mg/kg of APAP challenge caused acute liver damage. CAY10594 administration markedly blocked the acute liver injury in a dose-dependent manner, showing almost complete inhibition with 8 mg/kg of CAY10594. During the pathological progress of acute liver injury, GSH levels are decreased, and this is significantly recovered upon the administration of CAY10594 at 6 hours post APAP challenge. GSK-3β (Serine 9)/JNK phosphorylation is mainly involved in APAP-induced liver injury. CAY10594 administration strongly blocked GSK-3β (Serine 9)/JNK phosphorylation in the APAP-induced acute liver injury model. Consistently, sustained JNK activation in the cytosol and mitochondria from hepatocytes were also decreased in CAY10594-treated mice. Many types of immune cells are also implicated in APAP-induced liver injury. However, neutrophil and monocyte populations were not different between vehicle- and CAY10594-administered mice which are challenged with APAP. Therapeutic administration of CAY10594 also significantly attenuated liver damage caused by the APAP challenge, eliciting an enhanced survival rate. Taken together, these results indicate that PLD2 is involved in the intrinsic response pathway of hepatocytes driving the pathogenesis of APAP-induced acute liver injury, and PLD2 may therefore represent an important therapeutic target for patients with drug-induced liver injury.
Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease
Background. Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronically remittent and progressive inflammatory disorders. Phospholipase D2 (PLD2) is reported to be involved in the pathogenesis of several inflammatory diseases. However, the exact role of PLD2 in IBD is obscure. Methods. PLD2 expression was determined in peripheral blood cells and inflamed mucosa from patients with IBD by qRT-PCR. Colonic biopsies were also obtained from CD patients before and after infliximab (IFX) treatment to examine PLD2 expression. PLD2 selective inhibitor (CAY10594) was administrated daily by oral gavage in DSS-induced colitis mice. Bone marrow neutrophils from colitis mice were harvested to examine the migration using Transwell plate. Results. PLD2 was found to be significantly increased in peripheral blood cells and inflamed mucosa in patients with active IBD. Treatment with IFX could significantly decrease PLD2 expression in intestinal mucosa in patients with CD. Moreover, blockade of PLD2 with CAY10594 could markedly ameliorate DSS-induced colitis in mice and promote neutrophil migration. Conclusions. PLD2 plays a critical role in the pathogenesis of IBD. Blockade of PLD2 may serve as a new therapeutic approach for treatment of IBD.
CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation
The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium(+) uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.
Cucurbitacin B-, E-, and I-Induced Browning of White Adipocytes Is Promoted by the Inhibition of Phospholipase D2
The mechanism of white adipose tissue browning is not well understood; however, naturally occurring compounds are known to play a positive role. The effects of cucurbitacins B, E, and I on the browning of mature white adipocytes were investigated. First, the cell viability exhibited by cucurbitacins B, E, and I in pre- and mature adipocytes was verified. Cucurbitacins B, E, and I had no effect on cell viability in pre- and mature adipocytes at concentrations up to 300 nM. To investigate the characteristics of representative beige adipocytes, the formation and morphology of cucurbitacin B, E, and I lipid droplets were verified. The total lipid droplet surface area, maximum Feret diameter, and total Nile red staining intensity of cucurbitacin B-, E-, and I-treated adipocytes were lower than those of mature white adipocytes. Furthermore, treatment of white mature adipocytes with cucurbitacin B, E, and I led to the formation of several small lipid droplets that are readily available for energy expenditure. We evaluated the effect of cucurbitacins B, E, and I on the expression of representative browning markers UCP1, PGC1a, and PRDM16, which participate in the browning of white adipose tissue. Cucurbitacins B, E, and I increased the mRNA and protein expression levels of UCP1, PGC1a, and PRDM16 in a concentration-dependent manner. To promote energy consumption by beige adipocytes, active mitochondrial biogenesis is essential. Next, we investigated the effects of cucurbitacin B, E, and I on mitochondrial biogenesis in mature adipocytes. Mitochondrial mass increased when mature adipocytes were treated with cucurbitacin B, E, and I. The degree of cucurbitacin B-, E- and I-induced transformation of white adipocytes into beige adipocytes was in the order of Cu E > Cu B > Cu I. To verify the effect of phospholipase D2 on the browning of white adipocytes, CAY10594-a PLD2 pharmacological inhibitor, and a knockdown system were used. PLD2 inhibition and knockdown improved the expression levels of UCP1, PGC1a, and PRDM16. In addition, PLD2 inhibition and knockdown in mature white adipocytes promoted mitochondrial biosynthesis. The effect of PLD2 inhibition and knockdown on promoting browning of white adipocytes significantly increased when Cu B, Cu E, and Cu I were co-treated. These data indicate that mature white adipocytes' beige properties were induced by cucurbitacins B, E, and I. These effects became more potent by the inhibition of PLD2. These findings provide a model for determining anti-obesity agents that induce browning and increase energy expenditure in mature white adipocytes.
Activation of AMPK/TSC2/PLD by alcohol regulates mTORC1 and mTORC2 assembly in C2C12 myocytes
Background: Ethanol (EtOH) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (mTORC)1 and increased mTORC2 activities. In contrast, phospholipase D (PLD) and its metabolite phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less well defined. Herein, we examine the role that PLD and PA play in EtOH-mediated mTOR signaling.
Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50-minute incubation with PA in the presence or absence of EtOH. The phosphorylation state of various proteins was assessed by immunoblotting. Protein-protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit.
Results: PA levels and PLD activity increased in C2C12 myocytes exposed to EtOH (100 mM). Increased PLD activity was blocked by inhibitors of AMP-activated protein kinase (AMPK) (compound C) and phosphoinositide 3-kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented EtOH-induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14-3-3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in mTORC2 activity were not associated with altered binding of complex members and 14-3-3. PA increased S6K1 phosphorylation, with the associated increase in mTORC1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with mTOR, as well as suppression of mTORC2.
Conclusions: EtOH-induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. EtOH and PA regulate mTORC1 via a PI3K/AMPK/TSC2/PLD signaling cascade. PA stimulates mTORC1 function and suppresses activation of mTORC2 as part of an mTORC1/2 feedback loop.