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...
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| creator | Soares-Bezerra, Rômulo José Ferreira, Natiele Carla da Silva Alberto, Anael Viana Pinto Bonavita, André Gustavo Fidalgo-Neto, Antônio Augusto Calheiros, Andrea Surrage Frutuoso, Valber da Silva Alves, Luiz Anastacio |
| 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. |
| doi_str_mv | 10.1371/journal.pone.0123089 |
| format | Article |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0123089</identifier><identifier>PMID: 25993132</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adenosine Triphosphate - metabolism ; Alzheimer's disease ; Animals ; Arthritis ; ATP ; Cell Line ; Communication ; High-throughput screening ; High-Throughput Screening Assays ; Laboratories ; Leukemia ; Medical treatment ; Mice ; Natural products ; Neurodegenerative diseases ; Pharmaceutical industry ; Pharmaceutical sciences ; Pharmacology ; Physiology ; Purinergic P2X Receptor Antagonists - pharmacology ; R&D ; Research & development ; Rheumatoid arthritis ; Screening ; Spectrophotometry</subject><ispartof>PloS one, 2015-05, Vol.10 (5), p.e0123089-e0123089</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Soares-Bezerra et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Soares-Bezerra et al 2015 Soares-Bezerra et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c762t-c30d69e7709dab4129ce163363890ff2dafdf62c4194876b37cd4d9b800bc6343</citedby><cites>FETCH-LOGICAL-c762t-c30d69e7709dab4129ce163363890ff2dafdf62c4194876b37cd4d9b800bc6343</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437783/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437783/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25993132$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Soares-Bezerra, Rômulo José</creatorcontrib><creatorcontrib>Ferreira, Natiele Carla da Silva</creatorcontrib><creatorcontrib>Alberto, Anael Viana Pinto</creatorcontrib><creatorcontrib>Bonavita, André Gustavo</creatorcontrib><creatorcontrib>Fidalgo-Neto, Antônio Augusto</creatorcontrib><creatorcontrib>Calheiros, Andrea Surrage</creatorcontrib><creatorcontrib>Frutuoso, Valber da Silva</creatorcontrib><creatorcontrib>Alves, Luiz Anastacio</creatorcontrib><title>An Improved Method for P2X7R Antagonist Screening</title><title>PloS one</title><addtitle>PLoS One</addtitle><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.</description><subject>Adenosine Triphosphate - metabolism</subject><subject>Alzheimer's disease</subject><subject>Animals</subject><subject>Arthritis</subject><subject>ATP</subject><subject>Cell Line</subject><subject>Communication</subject><subject>High-throughput screening</subject><subject>High-Throughput Screening Assays</subject><subject>Laboratories</subject><subject>Leukemia</subject><subject>Medical treatment</subject><subject>Mice</subject><subject>Natural products</subject><subject>Neurodegenerative diseases</subject><subject>Pharmaceutical industry</subject><subject>Pharmaceutical sciences</subject><subject>Pharmacology</subject><subject>Physiology</subject><subject>Purinergic P2X Receptor Antagonists - pharmacology</subject><subject>R&D</subject><subject>Research & development</subject><subject>Rheumatoid arthritis</subject><subject>Screening</subject><subject>Spectrophotometry</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl1rFDEUhgdRbK3-A9EBQfRi13xNPm6EpVRdqFRaFe9CJh-zs8wma5Ip9t87252WHemF5CLh5DnvyTl5i-IlBHOIGfywDn30qptvg7dzABEGXDwqjqHAaEYRwI8PzkfFs5TWAFSYU_q0OEKVEBhidFzAhS-Xm20M19aUX21eBVO6EMtv6Be7LBc-qyb4NuXySkdrfeub58UTp7pkX4z7SfHj09n30y-z84vPy9PF-UwzivJMY2CosIwBYVRNIBLaQooxxVwA55BRzjiKNIGCcEZrzLQhRtQcgFpTTPBJ8Xqvu-1CkmO3SULKoYCIETQQyz1hglrLbWw3Kt7IoFp5GwixkSrmVndWYlI5pRElprakgqYWyHFec6ogoEyxQevjWK2vN9Zo63NU3UR0euPblWzCtSQEM8bxIPBuFIjhd29Tlps2adt1ytvQ374bcU4Zrgb0zT_ow92NVKOGBlrvwlBX70TlgsBK8IpWu7LzB6hhGbtp9WAN1w7xScL7ScLAZPsnN6pPSS6vLv-fvfg5Zd8esCururxKoetzG3yagmQP6hhSitbdDxkCuXP23TTkztlydPaQ9urwg-6T7qyM_wJNRvBS</recordid><startdate>20150519</startdate><enddate>20150519</enddate><creator>Soares-Bezerra, Rômulo José</creator><creator>Ferreira, Natiele Carla da Silva</creator><creator>Alberto, Anael Viana Pinto</creator><creator>Bonavita, André Gustavo</creator><creator>Fidalgo-Neto, Antônio Augusto</creator><creator>Calheiros, Andrea Surrage</creator><creator>Frutuoso, Valber da Silva</creator><creator>Alves, Luiz Anastacio</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20150519</creationdate><title>An Improved Method for P2X7R Antagonist Screening</title><author>Soares-Bezerra, Rômulo José ; Ferreira, Natiele Carla da Silva ; Alberto, Anael Viana Pinto ; Bonavita, André Gustavo ; Fidalgo-Neto, Antônio Augusto ; Calheiros, Andrea Surrage ; Frutuoso, Valber da Silva ; Alves, Luiz Anastacio</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c762t-c30d69e7709dab4129ce163363890ff2dafdf62c4194876b37cd4d9b800bc6343</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Adenosine Triphosphate - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Soares-Bezerra, Rômulo José</au><au>Ferreira, Natiele Carla da Silva</au><au>Alberto, Anael Viana Pinto</au><au>Bonavita, André Gustavo</au><au>Fidalgo-Neto, Antônio Augusto</au><au>Calheiros, Andrea Surrage</au><au>Frutuoso, Valber da Silva</au><au>Alves, Luiz Anastacio</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An Improved Method for P2X7R Antagonist Screening</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2015-05-19</date><risdate>2015</risdate><volume>10</volume><issue>5</issue><spage>e0123089</spage><epage>e0123089</epage><pages>e0123089-e0123089</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25993132</pmid><doi>10.1371/journal.pone.0123089</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Adenosine Triphosphate - metabolism Alzheimer's disease Animals Arthritis ATP Cell Line Communication High-throughput screening High-Throughput Screening Assays Laboratories Leukemia Medical treatment Mice Natural products Neurodegenerative diseases Pharmaceutical industry Pharmaceutical sciences Pharmacology Physiology Purinergic P2X Receptor Antagonists - pharmacology R&D Research & development Rheumatoid arthritis Screening Spectrophotometry |
| title | An Improved Method for P2X7R Antagonist Screening |
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