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

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