Kinase experiment:

Human astrocytoma cells, 1321N1, are grown to stably express rat P2X7, human P2X4, P2X2a, P2X2/3, P2X1, P2Y1 and P2Y2 recombinant receptors. Agonist, BzATP, 2,3-O-(4-ben-zoylbenzoyl)-ATP or ATP-induced changes in intracellular Ca2+ concentrations are assessed in all of the cell lines using the Ca2+ chelating dye, Fluo-4, in conjunction with a Fluorometric Imaging Plate Reader. The cells are plated out the day before the experiment onto poly-D-lysine-coated black 96 well plates. After the agonist addition, changes in intracellular Ca2+ concentrations are recorded, per second, for 3 min. Ligands are tested at 11 half-log concentrations from 10-10 to 10-4 M. BzATP or ATP concentrations corresponds to the EC70 values for each receptor to enable comparison of antagonist potencies across the multiple P2 receptor subtypes. A 438079 is added to the cell plate and fluorescence data are collected for 3 min before the addition of agonist, subsequently, data are then collected for another 2 min. The pEC50 or pIC50 values are derived from a single curve fit.

Animal experiment:

To confirm A 438079 reach the brain after systemic administration, P10 rat pups are injected with 5 mg/kg A 438079 and killed either 10 min, 30 min, or 2 h later (n=4 per group). Blood samples are centrifuged at 1000×g for 10 min to isolate the plasma. Samples are analyzed using liquid chromatography-mass spectrometry (LC-MS/MS) by a service provider. Briefly, protein is precipitated from 50 μL aliquots of the individual plasma or brain tissue homogenate, and A 438079 is quantified by LC-MS/MS from a five-point standard curve.

• Front Cell Dev Biol 8 (2020): 1352.

A438079 is a selective small-molecule antagonist of human P2X7 receptor with IC50 value of 300 nM [1].
The nucleotide receptor P2X7 receptor is a subtype of the P2X receptor family. It is expressed in cells of both the central nervous system and the immune system. Since the activation of P2X7 receptor by ATP is associated with the release of glutamate and cytokines, P2X7 receptor is thought to be a target of the treatment for inflammation and neurodegeneration. The tetrazole-based compound A438079 works as a competitive antagonist of P2X7 receptor and shows potent antinociceptive activity in rat model [2].
A 438079 was selective against P2X7 receptor and showed no significant activity against other P2X receptors including P2X2, P2X3 and P2X4 even at concentration up to 10 μM. For other cell-surface ion channels and receptors, A 438079 also showed no effect. In the FLIPR assay using human 1321N1 astrocytoma cells stably transfected with recombinant rat or human P2X7 receptors, pretreatment of A 438079 potently inhibited the calcium influx with IC50 values of 100 and 300 nM, respectively. In human THP-1 cells differentiated with IFNγ and LPS, A 438079 treatment inhibited interleukin-1β release stimulated by BzATP with pIC50 value of 6.7.Besides that, A 438079 was found to block the pore formation in humanTHP-1 cells [1 and 2].
In the rat model of neuropathic pain, administration of A 438079dose-dependently reduced the mechanical allodynia with ED50 value of 76 μM/kg. In mice with status epilepticus, administration of A 438079 resulted in decreased total seizure power, amplitude and reduced seizure behavior. In addition, A 438079 was found to attenuate acetaminophen-induced liver injury in mice [2, 3 and 4].
References:
[1] Donnelly-Roberts DL, Jarvis MF. Discovery of P2X7 receptor-selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states. Br J Pharmacol. 2007 Jul;151(5):571-9. Epub 2007 Apr 30.
[2] Nelson DW, Gregg RJ, Kort ME, Perez-Medrano A, Voight EA, Wang Y, Grayson G, Namovic MT, Donnelly-Roberts DL, Niforatos W, Honore P, Jarvis MF, Faltynek CR, Carroll WA. Structure-activity relationship studies on a series of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists. J Med Chem. 2006 Jun 15;49(12):3659-66.
[3] Jimenez-Pacheco A, Mesuret G, Sanz-Rodriguez A, Tanaka K, Mooney C, Conroy R, Miras-Portugal MT, Diaz-Hernandez M, Henshall DC, Engel T. Increased neocortical expression of the P2X7 receptor after status epilepticus and anticonvulsant effect of P2X7 receptor antagonist A-438079. Epilepsia. 2013 Sep;54(9):1551-61.
[4] Xie Y, Williams CD, McGill MR, Lebofsky M, Ramachandran A, Jaeschke H. Purinergic receptor antagonist A438079 protects against acetaminophen-induced liver injury by inhibiting p450 isoenzymes, not by inflammasome activation. Toxicol Sci. 2013 Jan;131(1):325-35.