Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y1 Receptor

The recent crystallization of the neuropeptide Y Y 1 receptor (Y 1 R) in complex with the argininamide-type Y 1 R selective antagonist UR-MK299 ( 2 ) opened up a new approach toward structure-based design of nonpeptidic Y 1 R ligands. We designed novel fluorescent probes showing excellent Y 1 R selectivity and, in contrast to previously described fluorescent Y 1 R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in 2 via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y 1 R binding affinities (p K i values of 9.36–9.95) and proved to be Y 1 R antagonists, as validated in a Fura-2 calcium assay. The versatile applicability of the probes as tool compounds was demonstrated by flow cytometry- and fluorescence anisotropy-based Y 1 R binding studies (saturation and competition binding and association and dissociation kinetics) as well as by widefield and total internal reflection fluorescence (TIRF) microscopy of live tumor cells, revealing that fluorescence was mainly localized at the plasma membrane.