P2Y1 Receptor (IC50 = 51.6 nM)

MRS2279 inhibits the production of phosphoinositides in turkey erythrocyte membranes when 2-MeSADP is added, with a pKb value of 7.75 [2]. In 1321N1 human astrocytoma cells, MRS2279 exhibits high-affinity competitive antagonism to the human P2Y1 receptor with a pKb value of 8.10 [2]. Human P2Y2, P2Y4, P2Y6, and P2Y11 receptor cognate agonist activation is unaffected by MRS2279, however it has a distinct effect on the P2Y1 receptor [2]. It has not been shown that MRS2279 can prevent the build-up of cyclic AMP by obstructing ADP via platelet Gi/adenylate cyclase-linked P2Y receptors [2].

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MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pKB=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y1 receptor (pKB=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y1 receptor since MRS2279 had no effect on activation of the human P2Y2, P2Y4, P2Y6, or P2Y11 receptors by their cognate agonists. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. In contrast, the P2Y1 receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affnity (pKB=8.05) similar to that observed at the human P2Y1 receptor heterologously expressed in 1321N1 cells. Taken together these results illustrate selective high affinity antagonism of the P2Y1 receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues [2].

In high-pressure ventilation mice, MRS2279 diammonium (2 μL, 1 nM; intracerebroventricular injection; 30 min before to mechanical ventilation) decreases mouse brain injury caused by mechanical ventilation[3].

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Inhibition of P2Y1 receptor activation ameliorated brain injury induced by mechanical ventilation in mice [3]
To validate whether mechanical ventilation induced brain injury by activating P2Y1 receptor in the mouse hippocampus, the experimental mice were intracerebroventricularly injected with the P2Y1 antagonist MRS2279 30 min prior to mechanical ventilation, using an injection of ACSF as control (Figure 5a). Mice exhibited shorter latency (HVT group: 63.61 ± 4.49 seconds; HVT + ACSF group: 64.25 ± 5.81 seconds, HVT + MRS2279 group: 37.17 ± 3.50 seconds) and swimming distances (HVT group: 756.53 ± 30.03 cm; HVT + ACSF group: 762.83 ± 38.06 cm, HVT + MRS2279 group: 559.0 ± 37.63 cm) and spent a longer period of time in the target quadrant (HVT group: 10.45 ± 0.72 seconds; HVT + ACSF group: 10.48 ± 0.67 seconds, HVT + MRS2279 group: 13.63 ± 0.54 seconds) after inhibiting P2Y1 receptor activation (p< 0.05, Figure 5b). Meanwhile, the number of neurons had increased (p< 0.05, Figure 5c), while the dysbindin-1 protein level was decreased in the hippocampus (p< 0.05, Figure 5d), and levels of TNF-α, IL-1β, IL-6, and DA were all decreased in the hippocampus (p< 0.05, Figure 5e). Overall, our findings elicited that inhibition of P2Y1 receptor activation attenuated mouse brain injury induced by mechanical ventilation.

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Blockage of DA receptor mitigated mouse brain injury induced by increased DA release mediated by mechanical ventilation via P2Y1 receptor activation [3]
Similarly, to validate whether P2Y1 receptor activation induced brain injury DA nerve conduction by increasing the DA expression pattern, the experimental mice were intraperitoneally injected with the DA receptor antagonist haloperidol 30 min before mechanical ventilation (Figure 5a). Similar to the function of MRS2279 , the mice injected with haloperidol showed vital improvements in cognition after mechanical ventilation (p< 0.05, Figure 6a), with shorter latency (HVT group: 63.61 ± 4.49 seconds; HVT + saline group: 62.71 ± 5.54 seconds, HVT + haloperidol group: 31.53 ± 4.82 seconds) and swimming distances (HVT group: 756.53 ± 30.03 cm; HVT + saline group: 753.46 ± 34.13 cm, HVT + haloperidol group: 565.85 ± 45.98 cm) and spent a longer period of time in the target quadrant (HVT group: 10.45 ± 0.72 seconds; HVT + saline group: 10.36 ± 0.63 seconds, HVT + haloperidol group: 12.34 ± 0.67 seconds). Meanwhile, the number of neurons had increased in the hippocampus (p< 0.05, Figure 6b), the levels of DA and dysbindin-1 protein showed no significant alterations (p> 0.05, Figure 6c-d), while the levels of TNF-α, IL-1β, and IL-6 were reduced (p< 0.05, Figure 6d). Collectively, our findings suggested that suppression of the DA receptor mitigated mouse brain injury induced by increased DA release mediated by mechanical ventilation via P2Y1 receptor activation.

Preparation of washed platelets [2]
Venous blood was obtained from healthy volunteers and mixed with 20% of the final volume of acid/citrate/dextrose. The blood was centrifuged 180×g for 20 min, and the platelet-rich plasma was removed and incubated for 1 h in the presence of 1 mM aspirin. The platelets were centrifuged at 1000×g and resuspended to a density of 2×108 platelets ml−1 in HEPES-buffered Tyrode solution containing 0.2% BSA and 0.05 U ml−1 apyrase.

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Assay of cyclic AMP accumulation in human platelets [2]
Cyclic AMP accumulation was measured as described previously (Meeker & Harden, 1982). Briefly, platelets isolated from 50 ml of blood were labelled with 1 μCi ml−1 [3H]-adenine for 1 h at 37°C. The platelets were then washed and resuspended in (mM): NaCl 137 , KCl 2.7, MgCl2 1, NaH2PO4 3, glucose 5 and HEPES 10, pH 7.4. Incubations were for 10 min in the presence of 200 μM 3-isobutyl-1-methyl xanthine, and the reaction was stopped with 10% trichloroacetic acid. [3H]-Cyclic AMP was quantitated after chromatography over Dowex and alumina columns.

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Platelet aggregation [2]
Platelet aggregation was measured using a four-channel Chrono-Log aggregometer . Briefly, a 500 μl aliquot of washed platelets, supplemented with 2 mM CaCl2 and 1 mg ml−1 fibrinogen, was stirred at 37°C and the indicated concentrations of ADP were added and aggregation monitored during an 8 min incubation. Antagonist effects of MRS2279 were studied by preincubating platelets for 2 min with the P2Y1 antagonist prior to addition of ADP. The baseline for the aggregation response was set using 500 μl of HEPES-buffered Tyrode solution.

Assay of P2Y1 receptor-promoted inositol lipid hydrolysis in turkey erythrocyte membranes [2]
P2Y1-receptor-promoted activation of phospholipase C was studied in turkey erythrocyte membranes as we have described (Boyer et al., 1989, 1996a). Briefly, erythrocytes were incubated in inositol-free medium with 0.5 mCi of 2-[3H]-myo-inositol (20 Ci/mmol) for 18 – 24 h in a humidified atmosphere of 95% air/5% CO2 at 37°C. Membranes were prepared and phospholipase C activity was measured in 25 μl of 3H-inositol-labelled membranes (approximately 175 μg of protein, 200 – 500,000 c.p.m. per assay) in a medium containing (mM): CaCl2 0.424, MgSO4 0.91, EGTA 2, KCl 115, KH2PO4 5, and HEPES 10, pH 7.0. Assays (200 μl final volume) contained 1 μM GTPγS and the indicated concentrations of nucleotide analogues. Membranes were incubated at 30°C for 5 min, and total [3H]-inositol phosphates were quantified by anion-exchange chromatography. Typical values for inositol phosphate accumulation were approximately 200 – 300 c.p.m. (basal), 2000 – 3000 c.p.m. (1 μM GTPγS alone), and 15,000 – 20,000 c.p.m. (30 nM 2MeSADP+1 μM GTPγS). The range of triplicate values was within 10% of the mean.

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Assay of inositol phosphate accumulation in P2Y receptor-expressing 1321N1 cells [2]
The P2Y1, P2Y2, P2Y4, P2Y6, or P2Y11 receptors were stably expressed in 1321N1 human astrocytoma cells using retroviral vectors as previously described (Schachter et al., 1996). Cells were labelled overnight with [3H]-inositol and agonist-promoted [3H]-inositol phosphate accumulation was quantitated after a 10 min incubation in the presence of 10 mM LiCl and the cognate agonist for each receptor. The agonists used were 2MeSADP or ADP for P2Y1 receptor expressing cells, UTP for P2Y2 receptor-expressing cells, UTP for P2Y4 receptor-expressing cells, UDP for P2Y6 receptor-expressing cells, and ATP for P2Y11 receptor-expressing cells. Typical values for inositol phosphate accumulation were 500 – 700 c.p.m. for basal and 4000 – 10,000 c.p.m. for accumulation in the presence of a maximally effective concentration of cognate agonist for each receptor. The range of triplicate values was within 10% of the mean.

C57BL6 mice aged 8–12 weeks were housed in specific pathogen-free animal rooms with ad libitum access to food and water under 12/12 h light-dark cycles. The spontaneously breathing mice (sham group) received the same sedation as the mice in other groups: low-pressure ventilation (LVT group) [peak inspiratory pressure (PIP) of 12 cm H2O; positive end-expiratory pressure of 2 cm H2O; respiratory rate of 100 breaths/min] or high-pressure ventilation (HVT group) (PIP of 20 cm H2O; positive end-expiratory pressure of 0 cm H2O; respiratory rate of 50 breaths/min) for 90 min, followed by an array of 330-min long-term ventilation experiments under high-pressure ventilation. High-pressure ventilated mice were randomly selected and intraperitoneally injected with the DA receptor antagonist haloperidol (0.5 mg/kg in 0.2 mL saline) 30 min prior to mechanical ventilation with the mice injected with an equivalent amount of normal saline as controls, or simultaneously intracerebroventricularly (coordinates with respect to bregma: AP = 0.4 mm; L = 0.95 mm) injected with 2 μL of the P2Y1 receptor antagonist MRS2279 (1 nmol, 96% purity; Tocris Bioscience, Abingdon, UK) 30 min prior to mechanical ventilation with mice injected with an equivalent amount of artificial cerebrospinal fluid (ACSF) as controls. Animals were assigned into the following groups with 12 mice in each group (total 96): 1. the sham group, spontaneous breathing; 2. the LVT group, low tidal volume; 3. the HVT group, high tidal volume; 4. the long term group, mechanical ventilation for 330 min under high tidal volume; 5. the HVT + ACSF group, high tidal volume mechanical ventilation was performed 30 min after intracerebroventricular injection of ACSF; 6. the HVT + MRS2279 group, high tidal volume mechanical ventilation was performed 30 min after lateral ventricular injection of MRS2279 ; 7. the HVT + saline group, normal saline was injected intraperitoneally 30 min before mechanical ventilation; 8. the HVT + haloperidol group, haloperidol was injected intraperitoneally 30 min before mechanical ventilation. All ventilated mice were euthanized (intraperitoneal administration of 200 mg/kg pentobarbital sodium) after conducting the Morris water maze test. Hippocampus and lung tissues of mice were harvested for subsequent experimentation. The tissues were randomly selected from 6 mice in each group for pathological examination while the tissues of the remaining mice were homogenized for protein expression detection.[3]

[1]. Synthesis, biological activity, and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y(1) receptor ligands. J Med Chem. 2000;43(5):829-842.

[2]. Boyer JL, et al, Ravi RG, Jacobson KA, Harden TK. 2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist. Br J Pharmacol. 2002;135(8):2004-2010.

[3]. Mechanical ventilation induces lung and brain injury through ATP production, P2Y1 receptor activation and dopamine release. Bioengineered. 2022 Feb;13(2):2346-2359.

The structure-activity relationships of adenosine-3', 5'-bisphosphates as P2Y(1) receptor antagonists have been explored, revealing the potency-enhancing effects of the N(6)-methyl group and the ability to substitute the ribose moiety (Nandanan et al. J. Med. Chem. 1999, 42, 1625-1638). We have introduced constrained carbocyclic rings (to explore the role of sugar puckering), non-glycosyl bonds to the adenine moiety, and a phosphate group shift. The biological activity of each analogue at P2Y(1) receptors was characterized by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit its stimulation elicited by 30 nM 2-methylthioadenosine-5'-diphosphate (antagonist effect). Addition of the N(6)-methyl group in several cases converted pure agonists to antagonists. A carbocyclic N(6)-methyl-2'-deoxyadenosine bisphosphate analogue was a pure P2Y(1) receptor antagonist and equipotent to the ribose analogue (MRS 2179). In the series of ring-constrained methanocarba derivatives where a fused cyclopropane moiety constrained the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle, the 6-NH(2) (N)-analogue was a pure agonist of EC(50) 155 nM and 86-fold more potent than the corresponding (S)-isomer. The 2-chloro-N(6)-methyl-(N)-methanocarba analogue was an antagonist of IC(50) 51.6 nM. Thus, the ribose ring (N)-conformation appeared to be favored in recognition at P2Y(1) receptors. A cyclobutyl analogue was an antagonist with IC(50) of 805 nM, while morpholine ring-containing analogues were nearly inactive. Anhydrohexitol ring-modified bisphosphate derivatives displayed micromolar potency as agonists (6-NH(2)) or antagonists (N(6)-methyl). A molecular model of the energy-minimized structures of the potent antagonists suggested that the two phosphate groups may occupy common regions. The (N)- and (S)-methanocarba agonist analogues were docked into the putative binding site of the previously reported P2Y(1) receptor model.[1]

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Mechanical ventilation can induce lung injury and exacerbate brain injury due to lung-brain interaction. The current study sought to investigate the mechanism of lung-brain interaction induced by mechanical ventilation and offer theoretical insight into the management of ventilator-induced brain injury. The experimental mice were assigned into the spontaneously breathing group and the mechanical ventilation group and injected with dopamine (DA) receptor antagonist haloperidol or P2Y1 receptor antagonist MRS2279 before ventilation. In vitro assay was conducted using lung epithelial cells MLE-12 hippocampal neuron cells and HT-22. Mouse recognition function and lung injury were examined. The condition and concentration of neurons in the hippocampus were observed. The levels of several inflammatory factors, DA, adenosine triphosphate (ATP), P2Y1R, and dysbindin-1 were detected. Mechanical ventilation induced lung and brain injury in mice, manifested in increased inflammatory factors in the bronchoalveolar lavage fluid and hippocampus, prolonged escape latency, and swimming distance and time in the target quadrant with a weakened concentration of neurons in the hippocampus. Our results presented elevated ATP and P2Y1R expressions in the mechanically ventilated mice and stretched MLE-12 cells. The mechanically ventilated mice and P2Y1 receptor activator MRS2365-treated HT-22 cells presented with elevated levels of DA and dysbindin-1. Inactivation of P2Y1 receptor in the hippocampus or blockage of DA receptor alleviated brain injury induced by mechanical ventilation in mice. To conclude, the current study elicited that lung injury induced by mechanical ventilation exacerbated brain injury in mice by increasing ATP production, activating the P2Y1 receptor, and thus promoting DA release.[3]

C13H20CLN5O8P2

469.71132

469.032

367909-40-8

MRS2279 diammonium;2387505-47-5

9847505

Typically exists as solid at room temperature

1.132

5

12

7

29

740

4

CNC1C2N=CN([C@@H]3[C@H]4C[C@@]4(COP(O)(O)=O)[C@@H](OP(O)(O)=O)C3)C=2N=C(Cl)N=1

LPZJKPSGEADHTQ-HLTSFMKQSA-N

InChI=1S/C13H20ClN5O8P2/c1-15-11-10-12(18-13(14)17-11)19(6-16-10)4-7-2-8(5-26-28(20,21)22)9(3-7)27-29(23,24)25/h6-9H,2-5H2,1H3,(H,15,17,18)(H2,20,21,22)(H2,23,24,25)/t7-,8-,9+/m1/s1

[(1S,2R,4R)-4-[(2-chloro-6-methylaminopurin-9-yl)methyl]-2-(phosphonooxymethyl)cyclopentyl] dihydrogen phosphate

MRS2279 MRS-2279 MRS 2279.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.)