title: 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 μM and 173-285 μM for ATP, BBG and OATP, respectively) [corrected].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.
authors.affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Rômulo José
authors.initials: RJ
authors.lastname: Soares-Bezerra
authors.affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Natiele Carla da Silva
authors.initials: NC
authors.lastname: Ferreira
authors.affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Anael Viana Pinto
authors.initials: AV
authors.lastname: Alberto
authors.affiliation: Laboratory of Integrated Research, Campus UFRJ-Macaé Professor Aloísio Teixeira, UFRJ, Rio de Janeiro, Brazil.
authors.forename: André Gustavo
authors.initials: AG
authors.lastname: Bonavita
authors.affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Antônio Augusto
authors.initials: AA
authors.lastname: Fidalgo-Neto
authors.affiliation: Laboratory of Immunopharmacology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Andrea Surrage
authors.initials: AS
authors.lastname: Calheiros
authors.affiliation: Laboratory of Immunopharmacology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Valber da Silva
authors.initials: Vda S
authors.lastname: Frutuoso
authors.affiliation: Laboratory of Cellular Communication, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors.forename: Luiz Anastacio
authors.initials: LA
authors.lastname: Alves
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D057166:High-Throughput Screening Assays; D051379:Mice; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology*
chemical_list: D058920:Purinergic P2X Receptor Antagonists; D000255:Adenosine Triphosphate