Covalent Inhibition of the Histamine H₃ Receptor

Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H₃ receptor (H₃R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H₃R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H₃R was validated with washout experiments and leads to inverse agonism on H₃R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H₃R conformation and to study the consequences of prolonged inhibition of the H₃R.