P2Y2 Receptor

ATP and UTP increased the intracellular glycogen content, enhanced the actin fiber stress response, and promoted the proliferation and migration of GC cells, while P2RY2 competitive antagonist AR-C118925XX reversed the changes induced by ATP. Knockdown of P2RY2 expression by shRNA inhibited the proliferation of GC cells. Activation of P2RY2 increased the expression of Snail, Vimentin, and β-catenin in GC cells, and down-regulated the expression of E-cadherin, while AR-C118925XX decreased the expression of these genes induced by ATP. Activation of P2RY2 activated AKT/GSK-3beta/VEGF signal to promote the proliferation of GC cells, and the P13/AKT signaling pathway LY294002 reversed the corresponding phenomenon, but no synergistic pharmacological properties of AR-C118925XX and LY294002 have been found. [2]

AR-C118925XX (ip) reduces the increase in skin thickness produced by bleomycin in mice [1].
The administration of P2Y2 receptor antagonist AR-C118925XX significantly inhibited bleomycin-induced dermal fibrosis in mice [1]
We found that the amount of ATP in the mouse skin was significantly enhanced by the injection of bleomycin for 7 days (Figure 6a), suggesting that bleomycin-induced skin injury and inflammation may enhance ATP release from various cells in the skin. Finally, we examined the effect of P2Y2 receptor antagonist AR-C118925XX injections on bleomycin-induced dermal fibrosis in mice. Mice with bleomycin-induced dermal fibrosis received intraperitoneal injections of AR-C118925XX or phosphate-buffered saline. Bleomycin-enhanced dermal thickness was significantly suppressed by AR-C118925XX injections (Figure 6b and c). We confirmed that bleomycin-induced dermal fibrosis, as shown by Masson-Trichrome staining, was significantly reduced by AR-C118925XX injections (Figure 6d). The number of α-smooth muscle actin+ myofibroblasts, CD68+ macrophages, and CD3+ T cells in lesional skin in bleomycin-treated mice was enhanced, and the numbers of bleomycin-induced myofibroblasts and macrophages in lesional skin were significantly inhibited by AR-C118925XX injections (Figure 6e). The number of bleomycin-induced T cells in lesional skin tended to be inhibited by AR-C118925XX injections, but this difference did not have statistical significance. These results suggest that bleomycin-induced ATP in the skin might be associated with skin fibrosis and that AR-C118925XX might have an inhibitory action on skin fibrosis in vivo.

Flu-3AM assay [2]
Calcium ion fluorescent probe was used to measure the change in free internal calcium concentration. AGS and HGC-27 cells were seeded in a 24-well plate, and the cells were treated with ATP (25, 50, and 100 μM), UTP (100 μM), AR-C118925XX (10 μM) and ATP + AR-C118925XX (10 μM) for 30 min. 300 µl 4 µM Fluo-3AM working solution was added and incubated at 37°C for 1 h and then cells were washed 3 times with PBS. Averaged Fluo-3 fluorescence signal was obtained from three separate experiments.

Real-time PCR analysis [1]
Human dermal fibroblasts were incubated in DMEM with or without ATP or adenosine at indicated concentrations for indicated times. Cells were pretreated with the nonselective P2 receptor antagonist suramin (100 μmol/L), P2X4 receptor antagonist 5-BDBD (100 μmol/L), P2X7 receptor antagonist AZ11645373 (1 μmol/L), P2Y1 receptor antagonist MRS2179 (100 μmol/L), P2Y2 receptor antagonists AR-C118925XX (10 μmol/L) and kaempferol (30 μmol/L), P2Y11 receptor antagonist NF157 (50 μmol/L), P2Y12 receptor antagonist clopidogrel (30 μmol/L), P2Y14 receptor antagonist PPTN hydrochloride (1 μmol/L), p38 inhibitor SB203580 (10 μmol/L), and BIRB796 (10 μmol/L) for 30 minutes and then stimulated with 1 mmol/L ATP for 1 hour.

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Western blot assay [1]
Western blot analyses were performed according to the previously described protocols (Motegi et al., 2011a, 2011b). To examine the effect of suramin, SB203580, AR-C118925XX, and kaempferol on the ATP-induced phosphorylation of p38 or collagen type I production, cells were pretreated with suramin (100 μmol/L), SB203580 (10 μmol/L), AR-C118925XX (10 μmol/L), kaempferol (10 nmol/L), or DMSO (vehicle control) for 30 minutes and then stimulated with ATP (1 mmol/L) for 1 hour, as well as stimulated with ATP and/or soluble IL-6 receptor (100 ng/ml) for 48 hours.

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PAS glycogen content detection [2]
AGS and HGC-27 cells planted in a 24-well plate were treated with or without ATP (100 μM), UTP (100 μM), AR-C118925XX (10 μM), ATP + AR-C118925XX (10 μM), ATP + LY294002 (20 mM), ATP + AR-C118925XX (10 μM) + LY294002 (20 mM) or shCon/shP2RY2 for 24 h. PAS fixative was added for 20 min and then was washed with PBS for 3 times. Oxidant was added for 15 min, and then was washed with PBS. Cells were washed twice with sodium sulfite solution for 2 min each time. Mayer hematoxylin dye solution was added for 2 min and then was washed with PBS. Then, the number of PAS staining positive cells was observed under an inverted microscope and was measured by using Image Pro Plus 6.0 software.

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Cell wound healing assay [2]
AGS and HGC-27 cells were inoculated in a 6-well plate. After the cells were evenly covered with the whole hole. scratches were made with the sterile 10 µl gun head. 1640 medium containing 10% FBS was added and cultured for 24 h. Cells were treated or untreated ATP (100 μM), UTP (100 μM), AR-C118925XX (10 μM), ATP + AR-C118925XX (10 μM), ATP + LY294002 (20mM), ATP + AR-C118925XX (10 μM) + LY294002 (20 mM), or shCon/shP2RY2. The pictures were taken under an inverted microscope at 0 and 24 h, and the percentage of wound healing between cell scratches was measured.

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Evaluation of cell migration ability [2]
The invasion ability of GC cells was analyzed with a 24-well Transwell chamber. The filter of the upper insert was added 200 µl serum-free cell suspension (2 × 104 cells), and the lower insert was added with 200 ul of 1640 medium containing 10% FBS. Cells were treated or untreated with ATP (100 μM), UTP (100 μM), AR-C118925XX (10 μM), ATP + AR-C118925XX (10 μM), ATP + LY294002 (20mM), or ATP + AR-C118925XX (10 μM) + LY294002 (20 mM) for 24 h, and the cells were fixed with 4% paraformaldehyde for 30 min. Subsequently, the cells were stained with crystal violet and counted in five random fields under the microscope.

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Actin fiber labeling assay [2]
AGS and HGC-27 cells seeded in 24-well plates were treated with ATP (100 μM) and ATP + AR-C118925XX (10 μM) for 24 h, and then were washed with PBS. Cells were fixed with 4% paraformaldehyde on ice for 15 min, and then were washed 3 times with PBS. 400 μl 0.5% Trion X-100 was added for 10 min and washed 3 times with PBS. 200 μl of PBS diluted with 3 μl of YF fluorescently labeled phalloidin solution was added and incubated for 30 min, and then washed with PBS. Then five visual fields were randomly selected under a laser confocal microscope (Lakar sp5) and photographed.

Evaluation of tumor growth by in vivo experiment [2]
BALB/c nude mice were reared in an aseptic environment controlled by light and temperature in the laboratory. AGS cells were collected and reconstituted in PBS (100 μl), and approximately 4 × 106 cells. Cells were injected subcutaneously into the lateral thigh of 4-week-old male nude mice, and the xenografts were allowed to grow. When the tumor was grown to 1 week later, the mice were randomly divided into three groups with six mice in each group. The PBS (control), ATP (100 µM), or ATP + AR-C118925XX (10 µM) were injected into xenotransplant tissue at twice a week. The tumor size was measured with Vernier calipers, induced tumor volume = [length × width2]/2 for about 1 month.

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Bleomycin-induced skin fibrosis model [1]
Dermal fibrosis was induced in 8-week-old C57BL/6 mice with injections of bleomycin. Injections of 300 μl of bleomycin at a concentration of 1 mg/ml were given five times per week for 2 weeks as previously described (Yokoyama et al., 2018). To examine the effect of AR-C118925XX , mice intraperitoneally received AR-C118925XX (7 mg/kg/day) dissolved in 100 μl of DMSO or DMSO alone five times per week for 2 weeks.

[1]. The Regulation of Skin Fibrosis in Systemic Sclerosis by Extracellular ATP via P2Y2 Purinergic Receptor. J Invest Dermatol. 2019 Apr;139(4):890-899.

[2]. AKT/GSK-3beta/VEGF signaling is involved in P2RY2 activation-induced the proliferation and metastasis of gastric cancer. Carcinogenesis. 2022 Dec 5:bgac095.

Tissue injury/hypoxia and oxidative stress induced-extracellular adenosine triphosphate (ATP) can act as damage-associated molecular pattern molecules, which initiate inflammatory response. Our objective was to elucidate the role of extracellular ATP in skin fibrosis in systemic sclerosis (SSc). We identified that hypoxia enhanced ATP release and that extracellular ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. There were no significant differences of P2X and P2Y receptor expression levels between normal and SSc fibroblasts. Nonselective P2 receptor antagonist and selective P2Y2 receptor antagonists, kaempferol and AR-C118925XX, significantly inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p38 inhibitors, SB203580, and doramapimod. Collagen type I production in SSc fibroblasts by ATP-induced IL-6/IL-6 receptor trans-signaling was inhibited by kaempferol and SB203580. The amount of ATP in bleomycin-treated skin was increased, and administration of AR-C118925XX significantly inhibited bleomycin-induced dermal fibrosis in mice. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc and that extracellular ATP-induced phosphorylation of p38 via P2Y2 receptor might enhance IL-6 and collagen type I production in SSc fibroblasts. P2Y2 receptor antagonist therapy could be a treatment for skin sclerosis in patients with SSc. [1]

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Studies have revealed the contribution of ATP-G-protein-coupled P2Y2 receptor (P2RY2) in tumor progression, but the role of P2RY2 in regulating the progression of gastric cancer (GC) and related molecular mechanisms are relatively lacking. Therefore, this study investigates the effects of P2RY2 on the proliferation and migration of GC through in vivo and in vitro experiments. The results showed that P2RY2 was expressed in GC tissues and GC cell lines. Adenosine triphosphate (ATP) increased the calcium influx in AGS and HGC-27 cells, and was dose-dependent with ATP concentration. ATP and UTP increased the intracellular glycogen content, enhanced the actin fiber stress response, and promoted the proliferation and migration of GC cells, while P2RY2 competitive antagonist AR-C118925XX reversed the changes induced by ATP. Knockdown of P2RY2 expression by shRNA inhibited the proliferation of GC cells. Activation of P2RY2 increased the expression of Snail, Vimentin, and β-catenin in GC cells, and down-regulated the expression of E-cadherin, while AR-C118925XX decreased the expression of these genes induced by ATP. Activation of P2RY2 activated AKT/GSK-3beta/VEGF signal to promote the proliferation of GC cells, and the P13/AKT signaling pathway LY294002 reversed the corresponding phenomenon, but no synergistic pharmacological properties of AR-C118925XX and LY294002 have been found. In vivo experiments showed that ATP-induced tumor growth, while AR-C118925XX inhibited ATP-induced tumor growth. Our conclusion is that P2RY2 activated the AKT/GSK-3beta/VEGF signal to promote the proliferation and migration of GC, suggesting that P2RY2 may be a new potential target for the treatment of GC.[2]

C28H23N7O3S

537.592323541641

537.158

C, 62.56; H, 4.31; N, 18.24; O, 8.93; S, 5.96

216657-60-2

54210200

Light yellow to yellow solid powder

1.5±0.1 g/cm3

1.756

4.78

3

7

5

39

1010

0

PVKNPGQAFNALOI-UHFFFAOYSA-N

InChI=1S/C28H23N7O3S/c1-15-3-8-20-17(11-15)5-6-18-12-16(2)4-9-21(18)24(20)22-14-35(28(37)30-26(22)39)13-19-7-10-23(38-19)25(36)29-27-31-33-34-32-27/h3-12,14,24H,13H2,1-2H3,(H,30,37,39)(H2,29,31,32,33,34,36)

5-[[5-(6,13-dimethyl-2-tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,9,12,14-heptaenyl)-2-oxo-4-sulfanylidenepyrimidin-1-yl]methyl]-N-(2H-tetrazol-5-yl)furan-2-carboxamide

AR-C118925XX; 216657-60-2; AR-C 118925XX; 5-[[5-(2,8-Dimethyl-5H-dibenzo[a,d]cyclohepten-5-yl)-3,4-dihydro-2-oxo-4-thioxo-1(2H)-pyrimidinyl]methyl]-N-2H-tetrazol-5-yl-2-furancarboxamide; CHEMBL4082045; 5-((5-(2,8-Dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide; 5-[(5-{6,13-dimethyltricyclo[9.4.0.0^{3,8}]pentadeca-1(11),3,5,7,9,12,14-heptaen-2-yl}-2-oxo-4-sulfanylidene-1,2,3,4-tetrahydropyrimidin-1-yl)methyl]-N-(1H-1,2,3,4-tetrazol-5-yl)furan-2-carboxamide; 5-[[5-(6,13-dimethyl-2-tricyclo[9.4.0.03,8]pentadeca-1(11),3(8),4,6,9,12,14-heptaenyl)-2-oxo-4-sulfanylidenepyrimidin-1-yl]methyl]-N-(2H-tetrazol-5-yl)furan-2-carboxamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~186.02 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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