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).
We synthesized D77, a novel tracer derived from the non-selective cannabinoid Δ8-THC. Using time-resolved Förster 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.
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.
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.
Copyright © 2025 Borrega-Roman, Hoare, Kosar, Sarott, Patej, Bouma, Scott-Dennis, Koers, Gazzi, Mach, Barrondo, Sallés, Guba, Kusznir, Nazaré, Rufer, Grether, Heitman, Carreira, Sykes and Veprintsev.

Cell surface density of G protein-coupled receptors (GPCRs) is tightly regulated through constitutive and agonist-induced internalization. Whereas the mechanisms of constitutive internalization remain elusive, agonist-induced internalization is accepted to involve receptor phosphorylation by GPCR kinases (GRKs), β-arrestin binding and AP2 recruitment, targeting receptors to clathrin-coated pits. Dimeric class C metabotropic glutamate (mGlu1 to 8) receptors regulate synaptic transmission but their internalization process is ambiguous. Here, we used diffusion-enhanced energy transfer (DERET) to decipher their internalization kinetics. We showed that all mGlu receptors are constitutively internalized. However, only mGlu1, 5 and 3 homodimers are agonist-induced internalized, that require neither GRKs, nor β-arrestins. In contrast, the constitutive internalization involves only β-arrestins. This systematic study further illustrates how different class C receptors are relative to most other GPCRs, revealing non-canonical internalization mechanisms. These insights in mGlu receptor dynamics will help promoting the therapeutic action of drugs targeting mGlu receptors.

The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.

INTRODUCTION The kinetics of ligand binding to G protein-coupled receptors (GPCRs) is an important determining factor in the preclinical evaluation of a molecule. Therefore, efforts should be made to measure this property as part of any drug development plan. The original assays used to assess ligand binding kinetics were developed using radioligands. However, these types of assays are very labor-intensive, limiting their application to the later phases of the drug discovery process. Recently, fluorescence-based ligand binding assays have been developed for multiple GPCRs, demonstrating their superiority through a homogeneous format and continuous data acquisition capabilities. The overriding aim of this study was to develop a fluorescence-based homogeneous ligand binding assay to profile the kinetics of compounds binding to human cannabinoid type 1 and 2 receptors (CB1R and CB2R). METHODS We designed and synthesized D77, a novel universal tracer based on the lower affinity non-selective naturally occurring psychoactive cannabinoid, Δ 8 -THC. Using the TR-FRET (time-resolved Förster resonance energy transfer) technique to develop an assay to study the kinetics of ligand binding to CB1R and CB2R at physiological temperature. To establish a CB1R construct suitable for this assay, it was necessary to truncate the first 90 amino acids of the flexible CB1R N-terminal domain, in order to reduce the FRET distance between the terbium cryptate (donor) and the fluorescent ligand (acceptor), while the full length CB2R construct remained functional due to its shorter N-terminus. We then used the Motulsky-Mahan competition binding model to study the binding kinetics of non-fluorescent ligands. RESULTS D77 tracer displayed affinity for the truncated human CB1R (CB1R 91-472 ) and full length CB2R (CB2R 1-360 ) in the nanomolar range, and competitive binding behavior with orthosteric ligands. Crucially, D77 displayed fast dissociation kinetics from both CB1R and CB2R, comparable to those of the most rapidly dissociating reference compounds tested. This unique property of D77 proved pivotal to accurately determining the on- and off-rates of the fastest dissociating compounds. Using D77, we successfully determined the kinetic binding properties of a series of CB1R and CB2R agonists and antagonists at 37°C, including rimonabant, which was marketed for the treatment of obesity but later withdrawn due to serious neurological side effects. DISCUSSION The k on values of molecules binding CB1R showed a difference of three orders of magnitude from the slowest associating compound, HU308 to the most rapid, rimonabant. Interestingly, we found a strong correlation between k on and affinity for compounds binding to CB1R, suggesting that the association rate is the main parameter determining the affinity of compounds binding to CB1R. For compounds binding to CB2R, both k on and k off parameters contributed as affinity determinants. However, in contrast to CB1R, a stronger correlation was found between the dissociation constant rate parameter and the affinity of these molecules, suggesting that a combination of k on and k off dictates the overall affinity of compounds binding to CB2R. Ultimately, exploring the kinetic parameters of potential cannabinoid drug candidates could help future drug development programs targeting these receptors.

The human ATP-binding cassette (ABC) transporter, ABCG2, is responsible for multidrug resistance in some tumours. Detailed knowledge of its activity is crucial for understanding drug transport and resistance in cancer, and has implications for wider pharmacokinetics. The binding of substrates and inhibitors is a key stage in the transport cycle of ABCG2. Here, we describe a novel binding assay using a high affinity fluorescent inhibitor based on Ko143 and time-resolved Förster resonance energy transfer (TR-FRET) to measure saturation binding to ABCG2. This binding is displaced by Ko143 and other known ABCG2 ligands, and is sensitive to the addition of AMP-PNP, a non-hydrolysable ATP analogue. This assay complements the arsenal of methods for determining drug:ABCG2 interactions and has the possibility of being adaptable for other multidrug pumps.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.