P2X7 receptor (Human recombinant P2X7 receptor: Negative allosteric modulator; pIC50 values ~6.9-7.2 at low agonist concentrations, varying with assay buffer and agonist)
P2X7 receptor (Rat recombinant P2X7 receptor: Positive allosteric modulator at higher concentrations (3-30 µM); shows modest inhibitory effect at low concentrations (0.1-1 µM))[1]

At the human P2X7 receptor, GW791343 diHClide (0.01, 0.03, 0.1, 0.3, 1, 3, 10 µM; 40 min) exhibits non-competitive antagonistic action [1]. On the human P2X7 receptor, GW791343 diHClide (3, 10, 30 µM; 40 minutes) has negative allosteric modulatory action [1]. SCN cells' ATP rhythms are improved by GW791343 diHClide (5 µM; 24-48 hours; ATP measured every 4 hours) [2].

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In HEK293 cells expressing human recombinant P2X7 receptors, GW791343 acted as a non-competitive antagonist, reducing maximal responses to ATP and BzATP in both NaCl and sucrose assay buffers. In NaCl buffer, it caused a slight decrease in agonist potency at 300-1000 nM. In sucrose buffer, it reduced maximal responses more markedly with ATP as agonist, and decreased BzATP potency at concentrations of 300 nM to 10 µM. The pIC50 values for GW791343 decreased as agonist concentration increased, and varied depending on the agonist and assay buffer used. At low agonist concentrations, pIC50 was approximately 6.9-7.2.[1]
At rat recombinant P2X7 receptors, the effects were species-specific. Low concentrations (100-1000 nM) of GW791343 slightly reduced responses to intermediate concentrations of BzATP in NaCl buffer, with no clear inhibitory effect when using ATP. The predominant effect at higher concentrations (3-30 µM) was to potentiate agonist-induced ethidium accumulation. In NaCl buffer, 10 and 30 µM GW791343 enhanced BzATP responses and increased the pEC50 and maximal response to ATP. In sucrose buffer, it also increased responses to ATP. At 4°C in sucrose buffer, GW791343 increased responses to both ATP and BzATP, even with a short 5-minute pre-exposure.[1]
Receptor protection and interaction studies demonstrated that GW791343 did not interact competitively at the ATP binding site (as decavanadate did not protect against its effects and it did not affect PPADS blockade) but did interact with the site labeled by compound-17. GW791343 prevented the slowly reversible blockade produced by compound-17 and reduced the potency of compound-17 in functional assays. Radioligand binding studies confirmed that GW791343 competitively inhibited the binding of [³H]-compound-17 to both human (pIC50 = 6.14 ± 0.09) and rat (pIC50 = 6.04 ± 0.10) P2X7 receptors.[1]

Radioligand binding assays were performed using membranes prepared from HEK293 cells expressing human or rat recombinant P2X7 receptors. The assay was conducted in a final volume of 200 µl of 50 mM Tris HCl buffer (pH 7.4) containing 0.01% bovine serum albumin. The radioligand [³H]-compound-17 was used at a concentration of 2-3 nM. Incubations were carried out for 60 minutes at room temperature. The reaction was terminated by vacuum filtration. Nonspecific binding was defined using 10 µM unlabeled compound-17. The ability of GW791343 to inhibit specific radioligand binding was assessed over a range of concentrations.[1]

Cell viability assay[1]
Cell Types: HEK293 cells (expressing human recombinant P2X7 receptor)
Tested Concentrations: 3, 10, 30 µM
Incubation Duration: 40 minutes (10 minutes of pre-incubation, 30 minutes of incubation with other P2X7 receptor antagonists)
Experimental Results: demonstrated slow reversal of human P2X7 receptor (full reversal after 45 minutes) with fast dissociation rate.

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Cell viability assay[2]
Cell Types: SCN cells (from 16 to 21 day old Wistar rats maintained under a controlled 12-12 hour light/dark cycle from birth)
Tested Concentrations: 5 µM (used every 4 hrs (hours) fresh medicated culture medium).
Incubation Duration: 24-48 hrs (hours) (ATP measured every 4 hrs (hours))
Experimental Results: The amplitude of ATP release rhythm and extracellular ATP accumulation was enhanced to 144% of the control level.

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Functional assays measured agonist-stimulated ethidium accumulation in HEK293 cells expressing rat or human recombinant P2X7 receptors. Cells were grown to confluence in 96-well plates pre-treated with poly-L-lysine. Before the assay, growth media was removed and cells were rinsed with assay buffer. Assay buffers contained (in mM): HEPES 10, N-methyl-D-glucamine 5, KCl 5.6, D-glucose 10, CaCl₂ 0.5 (pH 7.4), supplemented with either 280 mM sucrose (sucrose buffer) or 140 mM NaCl (NaCl buffer). Cells were incubated with GW791343 or vehicle for 40 minutes at room temperature (or as specified) before the addition of a mixture containing the agonist (ATP or BzATP) and ethidium bromide (100 µM final). Agonist incubation times varied: for human P2X7, 2 min in sucrose and 16 min in NaCl buffer; for rat P2X7, 1.5 min in sucrose and 8 min in NaCl buffer. Reactions were terminated by adding a stop solution containing reactive black 5, and cellular ethidium accumulation was immediately quantified by measuring fluorescence (excitation 530 nm, emission 620 nm) from below the plate using a fluorescence plate reader.[1]
For receptor protection studies to examine interactions between antagonists, cells were pre-incubated with the first antagonist (e.g., decavanadate or GW791343) for 10 minutes, followed by co-incubation with a second, more slowly reversible antagonist (e.g., PPADS or compound-17) for 30 minutes. Antagonists were then removed by extensive washing with assay buffer. After a specified washout period (e.g., 15 or 45 minutes), ATP-stimulated ethidium accumulation was measured as described above to assess the persistent inhibitory effects of the second antagonist in the presence or absence of the first.[1]
To study the dissociation of GW791343 from the human P2X7 receptor, cells were incubated with the antagonist for 40 minutes, followed by rapid washout. Cells were then incubated for varying times before a final challenge with ATP and ethidium to measure remaining inhibition.[1]

[1]. Negative and positive allosteric modulators of the P2X(7) receptor. Br J Pharmacol. 2008 Feb;153(4):737-50.

[2]. Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors. Front Pharmacol. 2018 Mar 6;9:192.

GW791343 (N²-(3,4-difluorophenyl)-N¹-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycine dihydrochloride) is a novel P2X7 receptor ligand. Its mechanism of action is complex and species-dependent. On the human P2X7 receptor, it acts as a negative allosteric regulator, producing a non-competitive antagonistic effect. It does not interact with the ATP-binding site, but rather binds to sites that overlap with or interact with the binding site of the negative allosteric regulator compound-17. On the rat P2X7 receptor, its main effect at high concentrations (3-30 µM) is positive allosteric regulation, enhancing the potency and efficacy of the agonist. This positive regulation is reversible and does not appear to be caused by nonspecific effects. The compound's ability to both inhibit (in humans, with a weaker effect in rats) and enhance (in rats) the function of the P2X7 receptor makes it a valuable pharmacological tool for studying receptor mechanisms and species differences in the pharmacology of the P2X7 receptor. [1]

C20H24N4OF2.HCL

410.8885

446.145

1019779-04-4

GW791343 trihydrochloride;309712-55-8

71576670

White to off-white solid powder

5.547

5

6

6

29

475

0

IYJPZTBIYHPSKF-UHFFFAOYSA-N

InChI=1S/C20H24F2N4O.2ClH/c1-14-2-3-15(13-26-8-6-23-7-9-26)10-19(14)25-20(27)12-24-16-4-5-17(21)18(22)11-16;;/h2-5,10-11,23-24H,6-9,12-13H2,1H3,(H,25,27);2*1H

2-(3,4-difluoroanilino)-N-[2-methyl-5-(piperazin-1-ylmethyl)phenyl]acetamide;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

H2O : ~100 mg/mL (~223.54 mM)
DMSO : ~20 mg/mL (~44.71 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 50 mg/mL (111.77 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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