Sotelo, E. | Search bioRxiv

bioRxiv2026

BRET-Based Ligand Binding at Recombinant and Endogenous Dopamine D2 Receptors

Dopamine D2 receptor (D2R) regulates diverse biological processes and is an important therapeutic target, underscoring the need for robust methods to quantify ligand binding at this receptor. Bioluminescence resonance energy transfer (BRET)-based ligand binding assays have emerged as a powerful platform for quantifying binding in living cells, but their application to D2R has been limited by the availability of suitable fluorescent tracers. Here, we developed three far-red fluorescent antagonists (CELT-240, CELT-383, and CELT-385) as BRET tracers for D2R. All three compounds produced specific, saturable D2R binding with nanomolar affinity. Further characterization of CELT-385 demonstrated its utility for kinetic and competition binding measurements and revealed high affinity binding at the closely related dopamine D3 receptor (D3R). We also extended BRET-based ligand binding beyond genetically-modified receptors using bystander BRET, enabling measurements at unmodified recombinant and endogenously expressed D2R. Together, these findings provide new tools for quantitative D2R/D3R ligand binding and a template for extending BRET-based ligand binding to endogenously expressed GPCRs.

pharmacology and toxicology↗

bioRxiv2026

Repurposing dopamine D3 receptor fluorescent ligands for D2 receptor

The dopamine D2 receptor is a principal target of antipsychotic and antiparkinsonian drugs, yet well-characterized fluorescent ligands for the D2 receptor remain scarce compared with its relative, the D3 receptor. Here we show that fluorescent ligands developed for the D3 subtype can be repurposed as tools for studying the D2 receptor pharmacology. Using a machine-learning-assisted, live-cell fluorescence microscopy assay, we screened twelve fluorescent probes and identified eight ligands that bound specifically to the D2 receptor at 10 nM. Four ligands exhibited classical saturable binding, yielding Kd in the nanomolar range, whereas the remaining probes displayed unsaturated, time-dependent binding profiles. Kinetic analyses revealed substantial differences in association and dissociation behavior, highlighting the importance of time-resolved measurements. Competition binding with dopamine and haloperidol yielded affinity estimates consistent with literature values. These results expand the toolkit of validated D2 receptor fluorescent probes and support cross-receptor repurposing as a strategy for fluorescent ligand development.

biochemistry↗

bioRxiv2021

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by Free Energy simulations

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications. Author SummaryThe design of new ligands as chemical modulators of G protein-coupled receptors (GPCRs) has benefited considerably during the last years of advances in both the structural and computational biology disciplines. Within the last, area, the use of free energy calculation methods has arisen as a computational tool to predict ligand affinities to explain structure-affinity relationships and guide lead optimization campaigns. However, our comprehension of the structural determinants of ligands with different pharmacological profile is scarce, and knowledge of the chemical modifications associated with an agonistic or antagonistic profile would be extremely valuable. We herein report an original implementation of the thermodynamic cycles associated with free energy perturbation (FEP) simulations, to mimic the conformational equilibrium between active and inactive GPCRs, and establish a framework to describe pharmacological profiles as a function of the ligands selectivity for a given receptor conformation. The advantage of this method resides into its simplicity of use, and the only consideration of active and inactive conformations of the receptor, with no simulation of the transitions between them. This model can accurately predict the pharmacological profile of series of full and partial agonists as opposed to antagonists of the A2A adenosine receptor, and moreover, how certain mutations associated with modulation of basal activity can influence this pharmacological profiles, which enables our understanding of such clinically relevant mutations.

biophysics↗