Article
An Improved Method for P2X7R Antagonist Screening
Registro en:
BEZERRA, Rômulo José Soares; et al. An Improved Method for P2X7R Antagonist Screening. Plos One, v.10, n.5, e0123089, 15p, May 2015.
1932-6203
10.1371/journal.pone.0123089
Autor
Bezerra, Rômulo José Soares
Ferreira, Natiele Carla da Silva
Alberto, Anael Viana Pinto
Binavita, André Gustavo
Fidalgo Neto, Antônio Augusto
Calgeiros, Andrea Surrage
Frutuoso, Valber da Silva
Alves, Luiz Anastácio
Resumen
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.