P2X7 Receptor

In a concentration-dependent manner, pre-incubation with A-804598 (0.1-10 μM; 1 hour) greatly reduces BzATP-induced cell loss. The highest protective effect against BzATP-induced cytotoxicity is shown by 3 μM A-804598[2].

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To complement the results of P2X7 knockdown, we sought to test the effect of a P2X7 antagonist (A-804598) in murine microglia. Primary microglia were incubated with varying concentrations of A-804598 for 1 h prior to exposure to BzATP. Cell viability was measured by CCK-8 assay, and microglial morphology was examined by confocal microscopy. As shown in Fig. 6a, BzATP alone resulted in approximately 40% microglial cell loss, while pre-incubation with A-804598 significantly attenuated BzATP-induced cell loss in a concentration-dependent manner. Three micromolar A-804598 exhibited the greatest protective effect against BzATP-induced cytotoxicity (Fig. 6b). Furthermore, we validated the protective effect of A-804598 in activated microglia. LPS-primed microglia were first exposed to varying concentrations of A-804598 and then treated with BzATP. As expected, the primed microglia exhibited amoeboid morphology and significant cell loss in response to BzATP (Fig. 6a). However, results of confocal microscopy and CCK-8 assay showed that cell loss by BzATP were counteracted by pre-incubation with A-804598 concentration dependently (Fig. 6c). Taken together, our findings revealed the protective effects of P2X7 antagonist A-804598 against BzATP-induced cytotoxicity in both inactivated and activated microglia, further demonstrating the mediating role of P2X7 in ATP-induced microglial cell death.[2]

In the lumbar spinal cord in the end stage of the disease, chroni therapy with A-804598 (intraperitoneal injection; 30 mg/kg; five times a week) reduces the expression of LC3B-II and SQSTM1/p62[3].

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It is known that the SQSTM1/p62 autophagy substrate accumulates, together with LC3B-II, in lumbar spinal cord of ALS mice during disease progression, when the autophagic flux is impaired (Zhang et al., 2011). We have therefore measured the level of LC3B-II and SQSTM1/p62 proteins in lumbar spinal cord of SOD1-G93A mice after having pharmacologically inhibited in vivo the P2X7 receptor by a chronic treatment in female SOD1-G93A mice with the blood-brain permeant A-804598, demonstrated to reach brain concentrations in rodents after oral or i.p. doses (Able et al., 2011; Iwata et al., 2016), administered at 30 mg/Kg from pre-onset to end stage of disease. We find that while the protein levels of LC3B-II (Figure 5A) and SQSTM1/p62 (Figure 5B) are confirmed to be both increased at end stage in vehicle-treated SOD1-G93A mice with respect to WT, in A-804598-treated ALS mice with respect to vehicle, the LC3B-II protein content appears unmodified, while SQSTM1/p62 is inhibited to basal levels (Figures 5A,B). As shown, neither behavioral scores (Figure 5C), disease onset (Figure 5D), nor survival (Figure 5E) are however affected by A-804598 when administered in female SOD1-G93A mice.[3]

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors[1].

Cell Cytotoxicity Assay[2]
Cell Types: microglial cell
Tested Concentrations: 0.1, 0.3, 1, 3, 10 μM
Incubation Duration: 1 hour
Experimental Results: Protected against BzATP-induced cytotoxicity in both inactivated and activated microglia.

Animal/Disease Models: Adult B6 .Cg-Tg (SOD1-G93A) 1Gur/J female mice [3]
Doses: 30 mg/kg
Route of Administration: intraperitoneal (ip)injection; five times a week
Experimental Results: diminished SQSTM1/p62 expression.

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SOD1-G93A mice at 100 days of age (pre-onset) were randomly grouped into vehicle-treated or CNS penetrant P2X7 specific antagonist A-804598-treated mice (Donnelly-Roberts et al., 2009; Catanzaro et al., 2014; Iwata et al., 2016) given by intraperitoneal injection at 30 mg/kg five times a week until end stage of disease. Because there is sex diversity in response to pharmacological treatments (Pizzasegola et al., 2009) and the P2X7 antagonist Brilliant Blue G has prolonged survival only in female SOD1-G93A mice (Bartlett et al., 2017; Sluyter et al., 2017), we have chosen to study female mice.[3]

[1]. [3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors. Neuropharmacology, 2009 Jan, 56(1):223-9.

[2]. The role of microglial P2X7: modulation of cell death and cytokine release. Neuroinflammation, 2017 Jul, 14(1):135.

[3]. P2X7 Receptor Activation Modulates Autophagy in SOD1-G93A Mouse Microglia. Front Cell Neurosci. 2017 Aug 21:11:249.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.[1]

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Background: ATP-gated P2X7 is a non-selective cation channel, which participates in a wide range of cellular functions as well as pathophysiological processes including neuropathic pain, immune response, and neuroinflammation. Despite its abundant expression in microglia, the role of P2X7 in neuroinflammation still remains unclear.
Methods: Primary microglia were isolated from cortices of P0-2 C57BL/6 wild-type or P2X7 knockout (P2X7-/-) mouse pups. Lipopolysaccharide, lipopolysaccharide plus IFNγ, or IL4 plus IL13 were used to polarize microglia to pro-inflammatory or anti-inflammatory states. P2rx7 expression level in resting or activated mouse and human microglia was measured by RNA-sequencing and quantitative real-time PCR. Microglial cell death was measured by cell counting kit-8 and immunocytochemistry, and microglial secretion in wild-type or P2X7-/- microglia was examined by Luminex multiplex assay or ELISA using P2X7 agonist BzATP or P2X7 antagonist A-804598. P2X7 signaling was analyzed by Western blot.
Results: First, we confirmed that P2rx7 is constitutively expressed in mouse and human primary microglia. Moreover, P2rx7 mRNA level was downregulated in mouse microglia under both pro- and anti-inflammatory conditions. Second, P2X7 agonist BzATP caused cell death of mouse microglia, while this effect was suppressed either by P2X7 knockout or by A-804598 under both basal and pro-inflammatory conditions, which suggests the mediating role of P2X7 in BzATP-induced microglial cell death. Third, BzATP-induced release of IL1 family cytokines including IL1α, IL1β, and IL18 was blocked in P2X7-/- microglia or by A-804598 in pro-inflammatory microglia, while the release of other cytokines/chemokines was independent of P2X7 activation. These findings support the specific role of P2X7 in IL1 family cytokine release. Finally, P2X7 activation was discovered to be linked to AKT and ERK pathways, which may be the underlying mechanism of P2X7 functions in microglia.
Conclusions: These results reveal that P2X7 mediates BzATP-induced microglial cell death and specific release of IL1 family cytokines, indicating the important role of P2X7 in neuroinflammation and implying the potential of targeting P2X7 for the treatment of neuroinflammatory disorders.[2]

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Autophagy and inflammation play determinant roles in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), an adult-onset neurodegenerative disease characterized by deterioration and final loss of upper and lower motor neurons (MN) priming microglia to sustain neuroinflammation and a vicious cycle of neurodegeneration. Given that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS, and that P2X7 affects autophagy in immune cells, we have investigated if autophagy can be directly triggered by P2X7 activation in primary microglia from superoxide dismutase 1 (SOD1)-G93A mice. We report that P2X7 enhances the expression of the autophagic marker microtubule-associated protein 1 light chain 3 (LC3)-II, via mTOR pathway and concomitantly with modulation of anti-inflammatory M2 microglia markers. We also demonstrate that the autophagic target SQSTM1/p62 is decreased in SOD1-G93A microglia after a short stimulation of P2X7, but increased after a sustained challenge. These effects are prevented by the P2X7 antagonist A-804598, and the autophagy/phosphoinositide-3-kinase inhibitor wortmannin (WM). Finally, a chronic in vivo treatment with A-804598 in SOD1-G93A mice decreases the expression of SQSTM1/p62 in lumbar spinal cord at end stage of disease. These data identify the modulation of the autophagic flux as a novel mechanism by which P2X7 activates ALS-microglia, to be considered for further investigations in ALS.[3]

C19H17N5

315.38

315.148

C, 72.36; H, 5.43; N, 22.21

1125758-85-1

53325874

White to off-white solid powder

4.298

2

3

5

24

473

1

N([H])(/C(/N([H])C#N)=N/[C@@]([H])(C([H])([H])[H])C1C([H])=C([H])C([H])=C([H])C=1[H])C1=C([H])C([H])=C([H])C2=C1C([H])=C([H])C([H])=N2

PQYCRDPLPKGSME-AWEZNQCLSA-N

InChI=1S/C19H17N5/c1-14(15-7-3-2-4-8-15)23-19(22-13-20)24-18-11-5-10-17-16(18)9-6-12-21-17/h2-12,14H,1H3,(H2,22,23,24)/t14-/m0/s1

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.93 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 25.0 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: 2.5 mg/mL (7.93 mM) 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; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (7.93 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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