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

The recent crystallization of the neuropeptide Y Y₁ receptor (Y₁R) in complex with the argininamide-type Y₁R selective antagonist UR-MK299 (<b>2</b>) opened up a new approach toward structure-based design of nonpeptidic Y₁R ligands. We designed novel fluorescent probes showing excellent Y₁R selectivity and, in contrast to previously described fluorescent Y₁R ligands, considerably higher (∼100-fold) binding affinity. This was achieved through the attachment of different fluorescent dyes to the diphenylacetyl moiety in <b>2</b> via an amine-functionalized linker. The fluorescent ligands exhibited picomolar Y₁R binding affinities (p<i>K</i>_i values of 9.36–9.95) and proved to be Y₁R 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 Y₁R 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.