Characterization of a selective and potent antagonist of human P2X₇ receptors, AZ11645373

Abstract

Background and purpose: 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. Experimental approach: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1b release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). Key results: AZ11645373 up to 10 mM, 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-1b release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC 50 ¼ 90 nM. AZ11645373 was 4 500-fold less effective at inhibiting rat P2X 7 receptor-mediated currents with less than 50% inhibition occurring at 10 mM. Conclusions and implications: 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.

The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro

“…1d). These compounds have been demonstrated to be potent, pIC 50 =7.3 for plasma membrane pore formation in THP-1 cells [22], and selective for P2X7R [23]. These results provide further evidence for the important role of the P2X7R in the formation of functional multinucleated human osteoclasts.…”

Purinergic and Store-Operated Ca2+ Signaling Mechanisms in Mesenchymal Stem Cells and Their Roles in ATP-Induced Stimulation of Cell Migration

“…ATPinduced increase in the [Ca 21 ] c was significantly reduced by 1 lM AZ11645373 (Fig. 2C), a human P2X7 selective antagonist [37]. In contrast, ATP-induced Ca 21 response was completely insensitive to 10 lM 5-BDBD (Fig.…”

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…More recently, Sharp et al reported that P2X 7 deficiency suppressed development of experimental autoimmune encephalomyelitis [44]. Therefore, P2X 7 receptors are promising targets in treatment of inflammatory diseases and pain regulation [45][46][47][48][49].…”

“…The ATP analog 2′,3′-O-(4-benzoyl-benzoyl) ATP (BzATP) is more potent and produces higher maximal responses [50]. P2X 7 receptor activity is blocked by divalent cations and certain antagonists such as oxidized ATP, KN-62, and AZ11645373 that are relatively selective for the receptor [47,51]. However, the pharmacological profile of these agonists and antagonists shows striking species differences among human, rat, mouse, and guinea pig orthologs [4,50,52].…”