Designing BRET-based conformational biosensors for G protein-coupled receptors
•Ligand-biased signaling will have significant impact on drug discovery programs.•Biosensor-based platforms have been developed to capture signaling signatures.•Signatures may be particular to cell types and thus not portable from cell to cell.•We capture receptor-proximal conformational profiles us...
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Veröffentlicht in: | Methods (San Diego, Calif.) Calif.), 2016-01, Vol.92, p.11-18 |
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creator | Sleno, Rory Pétrin, Darlaine Devost, Dominic Goupil, Eugénie Zhang, Alice Hébert, Terence E. |
description | •Ligand-biased signaling will have significant impact on drug discovery programs.•Biosensor-based platforms have been developed to capture signaling signatures.•Signatures may be particular to cell types and thus not portable from cell to cell.•We capture receptor-proximal conformational profiles using BRET-based sensors.•We discuss design/optimization of sensors for orthosteric and allosteric ligands.
Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands. |
doi_str_mv | 10.1016/j.ymeth.2015.05.003 |
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Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.</description><identifier>ISSN: 1046-2023</identifier><identifier>EISSN: 1095-9130</identifier><identifier>DOI: 10.1016/j.ymeth.2015.05.003</identifier><identifier>PMID: 25962643</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Adenosine A2 Receptor Antagonists - chemical synthesis ; Adenosine A2 Receptor Antagonists - metabolism ; Amino Acid Sequence ; Biased signaling ; Bioluminescence Resonance Energy Transfer Techniques - methods ; Biosensing Techniques - methods ; Biosensor ; Drug Design ; Fluorescent Dyes - chemical synthesis ; Fluorescent Dyes - metabolism ; G protein-coupled receptor ; HEK293 Cells ; Humans ; Luciferases, Renilla - chemical synthesis ; Luciferases, Renilla - metabolism ; Molecular Sequence Data ; Protein Structure, Secondary ; Receptor, Adenosine A2A - analysis ; Receptor, Adenosine A2A - metabolism ; Receptors, G-Protein-Coupled - chemistry ; Receptors, G-Protein-Coupled - genetics ; Receptors, G-Protein-Coupled - metabolism ; Resonance energy transfer ; Screening</subject><ispartof>Methods (San Diego, Calif.), 2016-01, Vol.92, p.11-18</ispartof><rights>2015 Elsevier Inc.</rights><rights>Copyright © 2015 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c429t-31ae13c2ee8cdd33c61b2481fa4d10ea09304448149b7231cc2eee494c7ff31e3</citedby><cites>FETCH-LOGICAL-c429t-31ae13c2ee8cdd33c61b2481fa4d10ea09304448149b7231cc2eee494c7ff31e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.ymeth.2015.05.003$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25962643$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sleno, Rory</creatorcontrib><creatorcontrib>Pétrin, Darlaine</creatorcontrib><creatorcontrib>Devost, Dominic</creatorcontrib><creatorcontrib>Goupil, Eugénie</creatorcontrib><creatorcontrib>Zhang, Alice</creatorcontrib><creatorcontrib>Hébert, Terence E.</creatorcontrib><title>Designing BRET-based conformational biosensors for G protein-coupled receptors</title><title>Methods (San Diego, Calif.)</title><addtitle>Methods</addtitle><description>•Ligand-biased signaling will have significant impact on drug discovery programs.•Biosensor-based platforms have been developed to capture signaling signatures.•Signatures may be particular to cell types and thus not portable from cell to cell.•We capture receptor-proximal conformational profiles using BRET-based sensors.•We discuss design/optimization of sensors for orthosteric and allosteric ligands.
Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.</description><subject>Adenosine A2 Receptor Antagonists - chemical synthesis</subject><subject>Adenosine A2 Receptor Antagonists - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Biased signaling</subject><subject>Bioluminescence Resonance Energy Transfer Techniques - methods</subject><subject>Biosensing Techniques - methods</subject><subject>Biosensor</subject><subject>Drug Design</subject><subject>Fluorescent Dyes - chemical synthesis</subject><subject>Fluorescent Dyes - metabolism</subject><subject>G protein-coupled receptor</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Luciferases, Renilla - chemical synthesis</subject><subject>Luciferases, Renilla - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Protein Structure, Secondary</subject><subject>Receptor, Adenosine A2A - analysis</subject><subject>Receptor, Adenosine A2A - metabolism</subject><subject>Receptors, G-Protein-Coupled - chemistry</subject><subject>Receptors, G-Protein-Coupled - genetics</subject><subject>Receptors, G-Protein-Coupled - metabolism</subject><subject>Resonance energy transfer</subject><subject>Screening</subject><issn>1046-2023</issn><issn>1095-9130</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kN9LwzAQgIMobk7_AkH66Evn5Ufb9cEHnXMKQ0Hmc0jT68xom5m0wv57Mzd9FA7uuHyXSz5CLimMKdD0Zj3eNth9jBnQZAwhgB-RIYU8iXPK4XhXizRmwPiAnHm_BgDKsskpGbAkT1kq-JC8PKA3q9a0q-j-bbaMC-WxjLRtK-sa1RnbqjoqjPXYeut8FNrRPNo426FpY237TR14hxo3XTg_JyeVqj1eHPKIvD_OltOnePE6f57eLWItWN7FnCqkXDPEiS5LznVKCyYmtFKipIAKcg5ChIbIi4xxqncoilzorKo4RT4i1_t7w0s-e_SdbIzXWNeqRdt7SbOEJZM0zdKA8j2qnfXeYSU3zjTKbSUFuRMp1_JHpNyJlBACeJi6OizoiwbLv5lfcwG43QMYvvll0EmvDbYaSxNsdLK05t8F34FIhaw</recordid><startdate>20160101</startdate><enddate>20160101</enddate><creator>Sleno, Rory</creator><creator>Pétrin, Darlaine</creator><creator>Devost, Dominic</creator><creator>Goupil, Eugénie</creator><creator>Zhang, Alice</creator><creator>Hébert, Terence E.</creator><general>Elsevier Inc</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>7X8</scope></search><sort><creationdate>20160101</creationdate><title>Designing BRET-based conformational biosensors for G protein-coupled receptors</title><author>Sleno, Rory ; Pétrin, Darlaine ; Devost, Dominic ; Goupil, Eugénie ; Zhang, Alice ; Hébert, Terence E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c429t-31ae13c2ee8cdd33c61b2481fa4d10ea09304448149b7231cc2eee494c7ff31e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Adenosine A2 Receptor Antagonists - chemical synthesis</topic><topic>Adenosine A2 Receptor Antagonists - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Biased signaling</topic><topic>Bioluminescence Resonance Energy Transfer Techniques - methods</topic><topic>Biosensing Techniques - methods</topic><topic>Biosensor</topic><topic>Drug Design</topic><topic>Fluorescent Dyes - chemical synthesis</topic><topic>Fluorescent Dyes - metabolism</topic><topic>G protein-coupled receptor</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Luciferases, Renilla - chemical synthesis</topic><topic>Luciferases, Renilla - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Protein Structure, Secondary</topic><topic>Receptor, Adenosine A2A - analysis</topic><topic>Receptor, Adenosine A2A - metabolism</topic><topic>Receptors, G-Protein-Coupled - chemistry</topic><topic>Receptors, G-Protein-Coupled - genetics</topic><topic>Receptors, G-Protein-Coupled - metabolism</topic><topic>Resonance energy transfer</topic><topic>Screening</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sleno, Rory</creatorcontrib><creatorcontrib>Pétrin, Darlaine</creatorcontrib><creatorcontrib>Devost, Dominic</creatorcontrib><creatorcontrib>Goupil, Eugénie</creatorcontrib><creatorcontrib>Zhang, Alice</creatorcontrib><creatorcontrib>Hébert, Terence E.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Methods (San Diego, Calif.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sleno, Rory</au><au>Pétrin, Darlaine</au><au>Devost, Dominic</au><au>Goupil, Eugénie</au><au>Zhang, Alice</au><au>Hébert, Terence E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Designing BRET-based conformational biosensors for G protein-coupled receptors</atitle><jtitle>Methods (San Diego, Calif.)</jtitle><addtitle>Methods</addtitle><date>2016-01-01</date><risdate>2016</risdate><volume>92</volume><spage>11</spage><epage>18</epage><pages>11-18</pages><issn>1046-2023</issn><eissn>1095-9130</eissn><abstract>•Ligand-biased signaling will have significant impact on drug discovery programs.•Biosensor-based platforms have been developed to capture signaling signatures.•Signatures may be particular to cell types and thus not portable from cell to cell.•We capture receptor-proximal conformational profiles using BRET-based sensors.•We discuss design/optimization of sensors for orthosteric and allosteric ligands.
Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>25962643</pmid><doi>10.1016/j.ymeth.2015.05.003</doi><tpages>8</tpages></addata></record> |
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subjects | Adenosine A2 Receptor Antagonists - chemical synthesis Adenosine A2 Receptor Antagonists - metabolism Amino Acid Sequence Biased signaling Bioluminescence Resonance Energy Transfer Techniques - methods Biosensing Techniques - methods Biosensor Drug Design Fluorescent Dyes - chemical synthesis Fluorescent Dyes - metabolism G protein-coupled receptor HEK293 Cells Humans Luciferases, Renilla - chemical synthesis Luciferases, Renilla - metabolism Molecular Sequence Data Protein Structure, Secondary Receptor, Adenosine A2A - analysis Receptor, Adenosine A2A - metabolism Receptors, G-Protein-Coupled - chemistry Receptors, G-Protein-Coupled - genetics Receptors, G-Protein-Coupled - metabolism Resonance energy transfer Screening |
title | Designing BRET-based conformational biosensors for G protein-coupled receptors |
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