An Improved Method for P2X7R Antagonist Screening.
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 upre...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4437783?pdf=render |
| _version_ | 1811205543985938432 |
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| author | Rômulo José Soares-Bezerra Natiele Carla da Silva Ferreira Anael Viana Pinto Alberto André Gustavo Bonavita Antônio Augusto Fidalgo-Neto Andrea Surrage Calheiros Valber da Silva Frutuoso Luiz Anastacio Alves |
| author_facet | Rômulo José Soares-Bezerra Natiele Carla da Silva Ferreira Anael Viana Pinto Alberto André Gustavo Bonavita Antônio Augusto Fidalgo-Neto Andrea Surrage Calheiros Valber da Silva Frutuoso Luiz Anastacio Alves |
| author_sort | Rômulo José Soares-Bezerra |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T03:34:17Z |
| format | Article |
| id | doaj.art-345fac264dbe451db92f88b86a1067a7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T03:34:17Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-345fac264dbe451db92f88b86a1067a72022-12-22T03:49:29ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01105e012308910.1371/journal.pone.0123089An Improved Method for P2X7R Antagonist Screening.Rômulo José Soares-BezerraNatiele Carla da Silva FerreiraAnael Viana Pinto AlbertoAndré Gustavo BonavitaAntônio Augusto Fidalgo-NetoAndrea Surrage CalheirosValber da Silva FrutuosoLuiz Anastacio AlvesATP 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.http://europepmc.org/articles/PMC4437783?pdf=render |
| spellingShingle | Rômulo José Soares-Bezerra Natiele Carla da Silva Ferreira Anael Viana Pinto Alberto André Gustavo Bonavita Antônio Augusto Fidalgo-Neto Andrea Surrage Calheiros Valber da Silva Frutuoso Luiz Anastacio Alves An Improved Method for P2X7R Antagonist Screening. PLoS ONE |
| title | An Improved Method for P2X7R Antagonist Screening. |
| title_full | An Improved Method for P2X7R Antagonist Screening. |
| title_fullStr | An Improved Method for P2X7R Antagonist Screening. |
| title_full_unstemmed | An Improved Method for P2X7R Antagonist Screening. |
| title_short | An Improved Method for P2X7R Antagonist Screening. |
| title_sort | improved method for p2x7r antagonist screening |
| url | http://europepmc.org/articles/PMC4437783?pdf=render |
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