Two-Step Ligand-Directed Covalent Fluorescent Labeling of the Adenosine A1-Receptor That Maintains Its Orthosteric Binding Site's Availability to Bind Ligands

J Med Chem. 2026 Feb 12;69(3):2481-2495. doi: 10.1021/acs.jmedchem.5c02389. Epub 2026 Jan 8.

Abstract

Genetic tagging of G protein-coupled receptors (GPCRs) with bioluminescent or fluorescent proteins is a well-established method for the study of ligand-binding and protein-protein interactions using resonance energy transfer approaches. Here we present a two-step, ligand-directed covalent labeling (LDCL) method that allows attachment of different fluorescent labels to an untagged adenosine A1 receptor using click chemistry. We also describe a range of biophysical approaches to confirm that the orthosteric binding site remains available to interact with endogenous ligands, agonists and antagonists, and access to the orthosteric binding site is not sterically hindered by the transferred cargo (fluorophore or click-reactive group).

MeSH terms

  • Animals
  • Binding Sites
  • Click Chemistry
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes* / chemistry
  • Fluorescent Dyes* / metabolism
  • Humans
  • Ligands
  • Protein Binding
  • Receptor, Adenosine A1* / chemistry
  • Receptor, Adenosine A1* / metabolism

Substances

  • Ligands
  • Fluorescent Dyes
  • Receptor, Adenosine A1