Development of covalent antagonists for ß1- and ß2-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 ß2-adrenergic receptor (ß2ARH2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic ß1- and ß2 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 ß1ARI2.64C and ß2ARH2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the ß2ARH2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the ß2ARH2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the ß1ARI2.64C and ß2ARH2.64C mutant while retaining a 41-fold selectivity for the ß1AR wild type over ß2AR. These compounds may serve as valuable molecular tools for studying ß1/ß2 subtype selectivity or investigations on GPCR trafficking and dimerization.

Potent haloperidol derivatives covalently binding to the dopamine D2 receptor.

The dopamine D2 receptor (D2R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D2R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D2R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments.