Publications

@article{Borrega-Roman2025-qf,

title = {A universal cannabinoid CB1 and CB2 receptor TR-FRET  kinetic ligand-binding assay},

author = {Leire Borrega-Roman and Bradley L Hoare and Miroslav Kosar and Roman C Sarott and Kacper J Patej and Jara Bouma and Morgan Scott-Dennis and Eline J Koers and Thais Gazzi and Leonard Mach and Sergio Barrondo and Joan Sall\'{e}s and Wolfgang Guba and Eric Kusznir and Marc Nazar\'{e} and Arne C Rufer and Uwe Grether and Laura H Heitman and Erick M Carreira and David A Sykes and Dmitry B Veprintsev},

doi = {10.3389/fphar.2025.1469986},

year  = {2025},

date = {2025-04-01},

urldate = {2025-04-01},

journal = {Front. Pharmacol.},

volume = {16},

pages = {1469986},

abstract = {Introduction: The kinetics of ligand binding to G protein-coupled 

 receptors (GPCRs) is an important optimization parameter in drug 

 discovery. Traditional radioligand assays are labor-intensive, 

 preventing their application at the early stages of drug 

 discovery. Fluorescence-based assays offer several advantages, 

 including a possibility to develop a homogeneous format, 

 continuous data collection, and higher throughput. This study 

 sought to develop a fluorescence-based binding assay to 

 investigate ligand-binding kinetics at human cannabinoid type 1 

 and 2 receptors (CB1R and CB2R). Methods: We synthesized D77, a 

 novel tracer derived from the non-selective cannabinoid 

 Δ8-THC. Using time-resolved F\"{o}rster resonance energy 

 transfer (TR-FRET), we developed an assay to study ligand-binding 

 kinetics at physiological temperatures. For CB1R, we truncated 

 the first 90 amino acids of its flexible N-terminal domain to 

 reduce the FRET distance between the terbium cryptate (donor) and 

 the fluorescent ligand (acceptor). The full-length CB2R construct 

 was functional without modification due to its shorter 

 N-terminus. The Motulsky-Mahan competition binding model was used 

 to analyze the binding kinetics of the endocannabinoids and 

 several other non-fluorescent ligands. Results: The D77 tracer 

 showed nanomolar-range affinity for truncated CB1R (CB1R91-472) 

 and full-length CB2R (CB2R1-360), displaying competitive binding 

 with orthosteric ligands. D77 exhibited rapid dissociation 

 kinetics from both CB1R and CB2R, which were similar to the 

 fastest dissociating reference compounds. This was critical for 

 accurately determining the on- and off-rates of the fastest 

 dissociating compounds. Using D77, we measured the kinetic 

 binding properties of various CB1R and CB2R agonists and 

 antagonists at physiological temperature and sodium ion 

 concentration. Discussion: The k on values for molecules binding 

 to CB1R varied by three orders of magnitude, from the slowest 

 (HU308) to the fastest (rimonabant). A strong correlation between 

 k on and affinity was observed for compounds binding to CB1R, 

 indicating that the association rate primarily determines their 

 affinity for CB1R. Unlike CB1R, a stronger correlation was found 

 between the dissociation rate constant k off and the affinity for 

 CB2R, suggesting that both k on and k off dictate the overall 

 affinity for CB2R. Exploring the kinetic parameters of 

 cannabinoid drug candidates could help drug development programs 

 targeting these receptors.},

keywords = {cannabinoid receptors, cannabinoid type 1, cannabinoid type 2, fluorescent ligand, kinetic ligand binding assay, ligand depletion, rebinding, time-resolved Forster resonance energy   transfer- based binding assay},

pubstate = {published},

tppubtype = {article}

}