An Improved Method for P2X7R Antagonist Screening

Abstract

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist

A new insight into purinergic pharmacology: Three fungal species as natural P2X7R antagonists

“…To identify extracts that inhibit the activation of P2X7R at a single established concentration (100 μg/mL), we applied the dye uptake methodology in the J774.G8 macrophage cell line. This methodology, previously described by our group, is based on the uptake of PI via the P2X7R pore when this receptor is activated by ATP at concentrations above 1 mM (Soares‐Bezerra et al, ). Using this method, we tested 1,800 plant and fungus extracts from several Brazilian and Antarctic biomes.…”

“…Because P2X7R activation by extracellular adenosine 5′‐triphosphate (ATP) in concentrations above 100 μM induces the opening of a nonselective pore that allows the passage of dyes, some HTS methodologies are based on this biophysical property (Namovic, Jarvis, & Donnelly‐Roberts, ). Thus, after performing an HTS campaign using a methodology previously described by our group (Soares‐Bezerra et al, ), 1,800 extracts were tested, and three of them significantly inhibited dye uptake. In this study, we described the potential antagonistic activity of these fungal extracts on the physiological roles of P2X7R in vitro .…”

Spergulin-A strives intra-macrophage leishmanicidal activity by regulating the host P2X7R-P38MAPK axis

“…Use of Brilliant Blue G (BB-G) as a selective P2X 7 R antagonist (Peng et al, 2009) was adequate in literature against both in vivo (Wang et al, 2017) and in vitro systems (Soares-Bezerra et al, 2015). Different IC50 values for different cell lines had been reported in the literature and also against a related mouse macrophage cell line (J774.G8) with applied dose range well within 10 μ M (Namovic et al, 2012;Soares-Bezerra et al, 2015), however not conclusive against RAW 264.7 MΦs. We evaluated the dose range for RAW 264.7 MΦ survival in accordance with the view of the dose range application for its inhibitory purposes against P2X 7 R (Figure.1b).…”

Molecular Structure and Regulation of P2X Receptors With a Special Emphasis on the Role of P2X2in the Auditory System

“…For P2X 2 , PPADS and TNP-ATP are active in low micromolar ranges (Jacobson et al, 2006). Brilliant Blue G (BBG) is one of the widely used P2X 7 receptor antagonist having an IC50 value of 10 nM (Jiang et al, 2000b;Lu et al, 2013;Boh ar et al, 2015;Nagahama et al, 2015;Soares-Bezerra et al, 2015).…”

“…At higher concentrations of agonists, 3.2-10 mM of ATP and 32-320 mM BzATP, a biphasic current was created, interpreted as the "dilated" state. This biphasic current had a higher permeability with an overall higher current production: (2015); Nagahama et al (2015); Soares-Bezerra et al (2015) the first phase was a rapid increase in current (I 1 ), followed by a second phase of a slower rate in rise in current (I 2 ) (Yan et al, 2008(Yan et al, , 2010. Studies have shown that the dilated pore can allow the passage of dyes and molecules with sizes up to 1.4 nm (Browne et al, 2013).…”