Different binding epitopes on the NK1 receptor for substance P and a non-peptide antagonist

NON-PEPTIDE ligands for peptide receptors have been discovered in several systems through file screening programs 1–6 , but the mechanism of action for these candidate drugs is obscure as they do not chemically resemble the native peptides. The compound CP 96345 is a high-affinity, non-peptide antagonist of the substance P (NK 1 ) receptor 4,5,7 , which is important in pain perception and neurogenic inflammation 8–11 . Here we identify epitopes on the NK 1 , receptor responsible for the specific binding of CP 96345 by systematic exchange of corresponding segments between the NK 1 , receptor and the homologous NK 3 (neurokinin B) receptor, which does not bind the non-peptide ligand. Non-conserved residues, in two epitopes around the top of transmembrane segment V and in one epitope at the top of transmembrane segment VI, are essential for the specific action of CP 96345 on the NK 1 , receptor, but are surprisingly not important for the binding of the natural peptide ligand, substance P. Susceptibility to the non-peptide antagonists can be conveyed to the previously unresponsive NK 3 receptor by mutational transfer of this discontinuous epitope from the NK 1 , receptor.