Structure-Based Design of High-Affinity Fluorescent Probes for the Neuropeptide Y Y-1 Receptor

The recent crystallization of the neuropeptide Y Y1receptor(Y1R) in complex with the argininamide-type Y1R selective antagonist UR-MK299 (2) opened up a new approach toward structure-based design ofnonpeptidic Y1R ligands. We designed novelfluorescent probes showingexcellent Y1R selectivity and, in contrast to previously describedfluorescentY1R ligands, considerably higher (similar to 100-fold) binding affinity. This wasachieved through the attachment of differentfluorescent dyes to thediphenylacetyl moiety in2via an amine-functionalized linker. Thefluorescent ligands exhibited picomolar Y1R binding affinities (pKivaluesof 9.36-9.95) and proved to be Y1R antagonists, as validated in a Fura-2calcium assay. The versatile applicability of the probes as tool compoundswas demonstrated by flow cytometry- and fluorescence anisotropy-basedY1R 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, evealing that fluorescence was mainly localized at the plasma membrane