Miroslav Kosar, Roman C. Sarott, David A. Sykes, Alexander E. G. Viray, Rosa Maria Vitale, Nataša Tomašević, Xiaoting Li, Rudolf L. Z. Ganzoni, Bilal Kicin, Lisa Reichert, Kacper J. Patej, Uxía Gómez-Bouzó, Wolfgang Guba, Peter J. McCormick, Tian Hua, Christian W. Gruber, Dmitry B. Veprintsev, James A. Frank*, Uwe Grether* and Erick M. Carreira*,
{"title":"Flipping the GPCR Switch: Structure-Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists","authors":"Miroslav Kosar, Roman C. Sarott, David A. Sykes, Alexander E. G. Viray, Rosa Maria Vitale, Nataša Tomašević, Xiaoting Li, Rudolf L. Z. Ganzoni, Bilal Kicin, Lisa Reichert, Kacper J. Patej, Uxía Gómez-Bouzó, Wolfgang Guba, Peter J. McCormick, Tian Hua, Christian W. Gruber, Dmitry B. Veprintsev, James A. Frank*, Uwe Grether* and Erick M. Carreira*, ","doi":"10.1021/acscentsci.3c01461","DOIUrl":null,"url":null,"abstract":"<p >We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB<sub>2</sub>R) selective inverse agonists (<i>S</i>)-<b>1</b> and (<i>R</i>)-<b>1</b>, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the <i>gem</i>-dimethylheptyl side chain. Epimer (<i>R</i>)-<b>1</b> exhibits high affinity for CB<sub>2</sub>R with <i>K</i><sub>d</sub> = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB<sub>2</sub>R independent of linker and fluorophore substitution. Ligands (<i>S</i>)-<b>1</b>, (<i>R</i>)-<b>1</b>, and their derivatives act as inverse agonists in CB<sub>2</sub>R-mediated cAMP as well as G protein recruitment assays and do not trigger β-arrestin–receptor association. Furthermore, no receptor activation was detected in live cell ERK<sub>1/2</sub> phosphorylation and Ca<sup>2+</sup>-release assays. Confocal fluorescence imaging experiments with (<i>R</i>)-<b>7</b> (Alexa488) and (<i>R</i>)-<b>9</b> (Alexa647) probes employing BV-2 microglial cells visualized CB<sub>2</sub>R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp258<sup>6.48</sup> and thereby stabilize CB<sub>2</sub>R in its inactive state.</p><p >We report a generalizable strategy for structure-based agonist-to-inverse-agonist functional transformation and probe development by ligand modification that modulates the GPCR toggle switch of CB<sub>2</sub>R.</p>","PeriodicalId":10,"journal":{"name":"ACS Central Science","volume":null,"pages":null},"PeriodicalIF":12.7000,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acscentsci.3c01461","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Central Science","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acscentsci.3c01461","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB2R) selective inverse agonists (S)-1 and (R)-1, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem-dimethylheptyl side chain. Epimer (R)-1 exhibits high affinity for CB2R with Kd = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-mediated cAMP as well as G protein recruitment assays and do not trigger β-arrestin–receptor association. Furthermore, no receptor activation was detected in live cell ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp2586.48 and thereby stabilize CB2R in its inactive state.
We report a generalizable strategy for structure-based agonist-to-inverse-agonist functional transformation and probe development by ligand modification that modulates the GPCR toggle switch of CB2R.
期刊介绍:
ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.