Development of covalent antagonists for β1- and β2-adrenergic receptors.

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β-adrenergic receptor (βAR) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β- and β receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants βAR and βAR. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the βAR within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the βAR at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the βAR and βAR mutant while retaining a 41-fold selectivity for the βAR wild type over βAR. These compounds may serve as valuable molecular tools for studying β/β subtype selectivity or investigations on GPCR trafficking and dimerization.