Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH₂ (2f-LI-) and Isox-Cha-Chg-Xaa-NH₂ (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the ¹⁹F-standard of an ¹⁸F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC₅₀ = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC₅₀ = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH₂ (EC₅₀ = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/μmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This ¹⁸F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.