Stabilized thioamide peptide agonists of the neuropeptide Y type 2 receptor for targeted cancer imaging

The neuropeptide Y type 2 receptor (Y₂R) is highly expressed in human neuroblastoma and glioblastoma cells, and it has been shown to stimulate cancer cell growth. To develop an effective imaging probe for glioblastomas, peptide-based agents can be designed as Y₂R agonists to be internalized by receptor-mediated endocytosis. However, the short half-life of most neuropeptides (<30 minutes) makes them unsuitable as imaging probes. Thioamide substitution, a single-atom O-to-S modification, is a promising tool to enhance peptide stability for therapeutic and imaging purposes. Herein, we designed and evaluated the first fluorescent cyclic thioamide peptides (HAP1 and HAP1-R^S ₃₃) as specific agonists and imaging agents of the Y₂R. High-performance liquid chromatography and mass spectrometry were used to identify cleavage sites by analyzing peptides after incubating in mouse serum, confirming enhanced stability of the peptides. Our stabilized cyclized thiopeptide probe showed a significant improvement in half-life from approximately 30 minutes to over 8 hours while maintaining moderate potency and high selectivity for binding with Y₂R receptor expressing cells, enabling selective imaging of Y₂R-expressing neuroblastoma cells. These results show that thioamide stabilized cyclized peptide probes targeting specific receptors may have potential for use in different biological or clinical applications.