P2Y2 Receptor
MRS2768 tetrasodium salt specifically targets the P2Y2 receptor, a GPCR that is activated by ATP and UTP. The compound is a selective agonist for this receptor, with an EC50 of 1.89 uM. It displays no affinity for the human P2Y4 or P2Y6 receptors, making it a useful tool for studying the specific pharmacology of P2Y2. Activation of P2Y2 triggers downstream signaling cascades, including Gq-mediated phospholipase C activation.

MRS2768 (0.01-10000 μM; 24 hours) greatly boosts the proliferation of PANC-1 cells[1].

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Incubation of PANC-1 cells with UTP or MRS2768, a selective P2Y2 receptor agonist, resulted in a dose- and time-dependent increase of proliferation. The messenger RNA transcript and protein of P2Y2 receptor were expressed in PANC-1 cells. P2 receptor antagonist suramin and small interfering RNA against P2Y2 receptor significantly decreased the proliferative effect of UTP and MRS2768. Activation of P2Y2 receptor by UTP transduced to phospholipase C, inositol 1,4,5-triphosphate (IP3), and protein kinase C. Uridine triphosphate-induced proliferation was mediated by protein kinase D, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, phosphatidylinositol 3-kinase (PI3K), Akt, and phospholipase D. Uridine triphosphate increased phosphorylation of Akt through protein kinase C, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, and PI3K. Conclusions: Uridine triphosphate increases proliferation of human pancreatic duct epithelial cells by activation of P2Y2 receptor and PI3K/Akt pathway. This could be helpful for discovering the long-term roles of P2Y2 receptor in pancreatic cells. [1]

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Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y2R antagonist, AR-C118925 (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) [2].

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In cell-free assays, MRS2768 tetrasodium salt is a P2Y2 receptor agonist with an EC50 of 1.89 uM. The compound functions to activate the P2Y2 receptor. It is selective for P2Y2 over other P2Y family members. The compound has a protective effect on cardiomyocytes from ischemic damage in vitro. This is likely mediated through the activation of survival pathways downstream of the P2Y2 receptor, such as Akt or ERK signaling.

In mice, myocardial injury is reduced by pretreatment with MRS2768 (4.44 μg/kg iv). Heart muscle cells are shielded from ischemia injury in vivo by MRS2768[2].

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In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo [2].

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Assessment of LV function post-MI using echocardiography [2]
There were no significant differences between groups in the echocardiographic measurements of cardiac structure or function at baseline. Induction of MI resulted in a more pronounced increase of left ventricular systolic diameter compared with the MRS2768-pretreated mice (p < 0.05, Fig. 3a, b). The infarcted mice also demonstrated a significantly reduced FS (33.4 ± 2.7 %) compared to MRS2768-treated mice (40.0 ± 3.13 %), or sham mice (51.8 ± 1.7 %, Fig. 3c, p < 0.001). Representative M mode pictures of the echo measurements are presented in Fig. 3e. Ischemic damage was measured using several markers as follows.

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Biochemical marker of ischemic damage [2]
Troponin T: The level of Troponin T, a specific marker denoting damage in the heart muscle, was significantly elevated in mice post-MI (13.4 ± 1.4 ng/ml) compared with the sham group. MRS2768 pretreatment significantly lowered Troponin T levels in the serum (10.6 ± 0.8 ng/ml, p < 0.05, Fig. 4a).

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In vivo, MRS2768 tetrasodium salt has been shown to be effective. Pretreatment with a low dose of MRS2768 (4.44 ug/kg, intravenous) reduces myocardial damage in mice, demonstrating a protective effect on cardiomyocytes from ischemic damage. It reduces infarct size in models of myocardial ischemia-reperfusion injury. These data support the potential cardioprotective role of the P2Y2 receptor and provide a proof-of-concept for this compound as a therapeutic lead.

P2Y2 Receptor siRNA Inhibition Assay [1]
Small interfering RNA against human P2Y2 receptor was used. The cells were grown in 24-well or 96-well plates at 70% to 80% confluence overnight and then transfected with predesigned siRNA (50 nmol/L) using Lipofectamine RNAiMAX transfection reagent as instructed by the manufacturer. A no-target siRNA (scrambled siRNA) was used as a negative control. Forty-eight hours later, cells were treated with UTP or MRS2768. The proliferation was measured after an additional 24-hour incubation period. P2Y2 messenger RNA (mRNA) expression was detected by quantitative RT-PCR and/or Western blotting to demonstrate successful silencing of the mRNA. The siRNA decreased the mRNA expression of the P2Y2 receptor more than 80% in the PANC-1 cells.

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The specific protocol for evaluating P2Y2 binding uses a radioligand binding assay. Membranes from 1321N1 human astrocytoma cells stably expressing the human P2Y2 receptor are incubated with [35S]-dATPalphaS (a radiolabeled agonist) and increasing concentrations of unlabeled MRS2768 tetrasodium salt (1 nM to 100 uM) in assay buffer for 60 minutes at room temperature. Bound and free radioactivity are separated by rapid vacuum filtration through GF/B filters. Non-specific binding is determined in the presence of 100 uM ATP. Filters are washed, dried, and counted. Ki values are calculated using the Cheng-Prusoff equation.

Cell Proliferation Assay[1]
Cell Types: Human pancreatic duct epithelial cells PANC-1
Tested Concentrations: 0.01, 0.1, 1, 10, 100, 1000, 10000 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The effect on proliferation of PANC-1 cells was dependent on concentration (0.1 μM to 1 mM). The concentration that elicited a half-maximal response (EC50) in the stimulation of proliferation was 0.8±1.7 μM. Resulted in a dose- and time-dependent increase of proliferation in PANC-1 cells

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For in vitro cellular assays, primary neonatal rat cardiomyocytes are isolated and cultured. Cells are subjected to oxygen-glucose deprivation (OGD) for 2-4 hours to simulate ischemia, followed by reoxygenation for 24 hours. MRS2768 tetrasodium salt (0.1, 1, 10 uM) is added to the culture medium immediately before the OGD period. Cell viability is assessed using the MTT assay or LDH release assay. Apoptosis is measured by caspase-3/7 activity using a fluorogenic substrate. The protective effect of the P2Y2 agonist is calculated as the percentage reduction in cell death compared to the OGD-only control group.

Animal/Disease Models: Male wild-type mice (C57BL)[2]
Doses: 4.44 μg/ kg
Route of Administration: Injected iv; 1 h before myocardial infarct (MI)
Experimental Results: Pretreatment decreased myocardial damage. The damage was Dramatically smaller in mice compared to the untreated mice (25.6±4.5% vs. 39.2±6.3%).

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Animals and experimental protocol [2]
Male wild-type mice (C57BL) were used. Three experimental groups were used:
1. sham without ligation of the left anterior descending coronary artery (LAD)
2. myocardial infarction (MI) = LAD ligation
3. animals injected with MRS2768 (4.44 μg/kg i.v.) 1 h before MI

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A standard in vivo protocol for MRS2768 uses a mouse model of myocardial ischemia-reperfusion (I/R) injury. Male C57BL/6 mice (8-10 weeks old) are anesthetized and subjected to left anterior descending (LAD) coronary artery ligation for 30 minutes, followed by reperfusion for 24 hours. MRS2768 tetrasodium salt is administered as a single intravenous (i.v.) injection (via tail vein) at a dose of 4.44 ug/kg, given 5 minutes prior to LAD ligation. After 24 hours of reperfusion, hearts are excised. The area at risk (AAR) is determined by Evans Blue staining, and the infarct size is assessed by TTC (2,3,5-triphenyltetrazolium chloride) staining. Infarct size is calculated as a percentage of the AAR.

Detailed PK data for MRS2768 tetrasodium salt is not available. As a nucleotide analog (tetraphosphate), it is highly charged and is not expected to be orally bioavailable. It is typically administered intravenously for in vivo studies. In plasma, it may be subject to rapid degradation by ectonucleotidases and alkaline phosphatases, which would cleave the phosphate groups. Its short half-life is acceptable for acute cardioprotective interventions such as ischemia-reperfusion injury models.

Specific toxicity data for MRS2768 tetrasodium salt is not available. As a selective P2Y2 receptor agonist, chronic activation of P2Y2 could potentially have off-target effects, such as alterations in ion transport in epithelial tissues or immune modulation. Standard safety screening would involve cellular toxicity assays (e.g., MTT on primary hepatocytes) to determine the maximum non-toxic concentration. The compound is used at low microgram per kg doses, which are typically well-tolerated.

[1]. Uridine Triphosphate Increases Proliferation of Human Cancerous Pancreatic Duct Epithelial Cells by Activating P2Y2 Receptor. Pancreas. 2013 May;42(4):680-6.

[2]. P2Y2 Receptor Agonist With Enhanced Stability Protects the Heart From Ischemic Damage in Vitro and in Vivo. Purinergic Signal. 2013 Dec;9(4):633-42.

This study found that the P2Y2 receptor mediates the proliferation of UTP in PANC-1 cells. Urate triphosphate (UTP) is a potent P2Y2/P2Y4 receptor agonist, while MRS2768 is a selective P2Y2 receptor agonist; both regulate the proliferation of pancreatic ductal epithelial cells in a concentration-dependent manner. MRS2768 is more potent than UTP, indicating that UTP regulates cell proliferation by activating the P2Y2 receptor. Furthermore, the P2 receptor antagonist suramin significantly inhibited UTP- and MRS2768-induced cell proliferation. siRNA targeting the P2Y2 receptor also significantly inhibited UTP- and MRS2768-induced cell proliferation, further supporting the involvement of the P2Y2 receptor. Based on the expression level of P2Y receptor mRNA, the expression level of the P2Y2 receptor in pancreatic cells is higher than that of the P2Y1 and P2Y4 receptor subtypes. The regulation of cell proliferation by UTP is mediated by the P2Y2 receptor. Studies on P2Y2 receptor activation in ovarian cancer^15,36 and colorectal cancer^21,27 have shown that UTP promotes cell proliferation in some cell lines, while it inhibits cell proliferation in others. This may be due to differences in cancer cell lines, P2 receptor expression levels, and related signaling pathways. In addition, the pro-proliferative effect of UTP is serum-dependent, as UTP has no effect on pancreatic cell proliferation under serum-free conditions (data not shown)^[1]. Since no difference in IκB levels was observed between ischemic groups, we can infer that NFκB degradation is not one of the mechanisms by which MRS2768 exerts its cardioprotective effect. Regarding the activation of c-Jun N-terminal kinase (JNK), there are currently conflicting results. Some studies have shown that JNK activation specifically occurs during reperfusion. We and other researchers have found that ischemia alone can activate this pathway. Furthermore, the role of JNK in myocardial ischemia/reperfusion (I/R) remains controversial, as equally reliable studies have reported contradictory results, showing both cardioprotective and detrimental effects. Contrary to our previous report, we found that wild-type (WT) mice pretreated with UTP showed significantly reduced expression of phosphorylated c-Jun compared to mice with isolated myocardial infarction (MI) (p < 0.05). This difference is likely due to the different in vivo half-lives, i.e., UTP degrades faster than MRS2768. However, the in vivo pharmacokinetics and pharmacodynamics of MRS2768 remain to be determined. The cardioprotective effect of MRS2768 appears to be partly related to c-Jun phosphorylation. In summary, we have demonstrated that MRS2768 activation of the P2Y2 receptor protects the heart from ischemic injury, minimizes infarct size, and improves cardiac function after myocardial infarction. Although the exact mechanism of inhibiting myocardial ischemia injury remains unclear, therapeutic strategies targeting the P2Y receptor show great promise in preventing ischemic myocardial injury and warrant further investigation. [2]

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MRS2768 tetrasodium salt is a research-grade compound and is not approved for clinical use. Its molecular weight is 728.14, with a molecular formula of C15H16N2Na4O18P4 and a purity of ≥98% (HPLC). It is stored at -80degC. Its selectivity for P2Y2 over P2Y4 and P2Y6 makes it a unique pharmacological tool to study the specific role of P2Y2 in cardiovascular protection. It is an important compound for validating P2Y2 as a drug target for myocardial infarction.

C15H16N2NA4O18P4

728.14

727.893936

2567869-47-8

90488893

Colorless to light yellow liquid

3

18

12

43

1120

4

C1=CC=C(C=C1)OP(=O)([O-])OP(=O)([O-])OP(=O)([O-])OP(=O)([O-])OC[C@@H]2[C@H]([C@H]([C@@H](O2)N3C=CC(=O)NC3=O)O)O.[Na+].[Na+].[Na+].[Na+]

ASYFBNZFGLWLNC-YYXHNCPRSA-J

InChI=1S/C15H20N2O18P4.4Na/c18-11-6-7-17(15(21)16-11)14-13(20)12(19)10(31-14)8-30-36(22,23)33-38(26,27)35-39(28,29)34-37(24,25)32-9-4-2-1-3-5-9;;;;/h1-7,10,12-14,19-20H,8H2,(H,22,23)(H,24,25)(H,26,27)(H,28,29)(H,16,18,21);;;;/q;4*+1/p-4/t10-,12-,13-,14-;;;;/m1..../s1

tetrasodium;[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-oxidophosphoryl] [oxido-[oxido(phenoxy)phosphoryl]oxyphosphoryl] phosphate

MRS2768 tetrasodium salt; 2567869-47-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping with dry ice.

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)