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The neuropeptide Y (NPY) Y 4 receptor (Y 4 R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y 4 R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y 4 R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y 4 R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y 4 R ligands derived from recently reported cyclic hexapeptides showing picomolar Y 4 R binding affinity. With p K i values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands ( 16 - 19 ) showed excellent Y 4 R affinity. Y 4 R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y 4 R (equilibrium p K d : 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y 4 R competition binding studies and imaging techniques such as single-receptor molecule tracking., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Derivatives of the rhodamine-based dye 5-TAMRA (5-carboxy-tetramethylrhodamine) and the indocarbocyanine-type Cy3B (cyclized derivative of the cyanine dye Cy3), both representing important fluorophores frequently used for the labeling of biomolecules (proteins, nucleic acids) and bioactive compounds, such as receptor ligands, were photophysically investigated in aqueous solution, i.e., in neat phosphate-buffered saline (PBS) and in PBS supplemented with 1 wt % bovine serum albumin (BSA). The dyes exhibit comparable absorption (λ abs,max : 550-569 nm) and emission wavelengths (λ em,max : 580-582 nm), and similar S 1 lifetimes (2.27-2.75 ns), and their excited state deactivation proceeds mainly via the lowest excited singlet state (triplet quantum yield ca. 1%). However, the probes show marked differences with respect to their fluorescence quantum yield and photostability. While 5-TAMRA shows a lower quantum yield (37-39%) than the Cy3B derivative (ca. 57%), its photostability is considerably higher compared to Cy3B. Generally, the impact of the protein on the photophysics is low. However, on prolonged illumination, both fluorescent dyes undergo a photocatalytic reaction with tryptophan residues of BSA mediated by sensitized singlet oxygen resulting in a tryptophan photoproduct with an absorption maximum around 330 nm. The overall results of this work will assist in choosing the right dye for the labeling of bioactive compounds, and the study demonstrates that experiments performed with 5-TAMRA or Cy3B-labeled compounds in a biological environment may be influenced by photochemical modification of experimentally relevant proteins at aromatic amino acid residues.

The family of human neuropeptide Y receptors (YRs) comprises four subtypes (Y 1 R, Y 2 R, Y 4 R, and Y 5 R) that are involved in the regulation of numerous physiological processes. Until now, Y 4 R binding studies have been predominantly performed in hypotonic sodium-free buffers using 125 I-labeled derivatives of the endogenous YR agonists pancreatic polypeptide or peptide YY. A few tritium-labeled Y 4 R ligands have been reported; however, when used in buffers containing sodium at a physiological concentration, their Y 4 R affinities are insufficient. Based on the cyclic hexapeptide UR-AK86C, we developed a new tritium-labeled Y 4 R radioligand ([ 3 H]UR-JG102, [ 3 H] 20 ). In sodium-free buffer, [ 3 H] 20 exhibits a very low Y 4 R dissociation constant ( K d 0.012 nM). In sodium-containing buffer (137 mM Na + ), the Y 4 R affinity is lower ( K d 0.11 nM) but still considerably higher compared to previously reported tritiated Y 4 R ligands. Therefore, [ 3 H] 20 represents a useful tool compound for the determination of Y 4 R binding affinities under physiological-like conditions.

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.