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

Area of Science:

• Medicinal Chemistry
• Pharmacology
• Molecular Biology

Background:

• Neuropeptide Y Y1 receptor (Y1R) structure-based design is crucial for developing selective ligands.
• Previous fluorescent Y1R ligands lacked high binding affinity.
• Crystallization of Y1R with UR-MK299 provided a template for novel ligand design.

Purpose of the Study:

• To design and synthesize novel fluorescent probes with enhanced Y1R selectivity and binding affinity.
• To utilize structure-based design principles derived from Y1R-antagonist complex crystallization.
• To develop versatile tool compounds for Y1R research.

Main Methods:

• Modification of the Y1R antagonist UR-MK299 by attaching fluorescent dyes via an amine linker.
• Characterization of fluorescent ligand binding affinity using pKi values.
• Validation of antagonist activity using Fura-2 calcium assays.
• Application in flow cytometry, fluorescence anisotropy, and TIRF microscopy.

Main Results:

• Novel fluorescent probes demonstrated high Y1R selectivity and approximately 100-fold increased binding affinity compared to previous ligands.
• Picomolar Y1R binding affinities (pKi values 9.36-9.95) were achieved.
• Probes confirmed as Y1R antagonists in functional assays.
• Successful visualization of Y1R localization on live tumor cell plasma membranes.

Conclusions:

• The developed fluorescent probes are potent and selective Y1R antagonists.
• These probes serve as valuable tool compounds for studying Y1R in various biological contexts.
• The findings facilitate structure-based drug design for nonpeptidic Y1R ligands.