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Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373.

ABSTRACT: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized.We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP).AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM.AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.

SUBMITTER: Stokes L

PROVIDER: S-EPMC2014691 | biostudies-other | 2006 Dec

REPOSITORIES: biostudies-other

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Project description:RationaleThe role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission.Experimental approachLSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs).ResultsSynaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R.ConclusionsPurines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

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Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373.

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