Lanthanide labeling of a potent protease activated receptor-2 agonist for time-resolved fluorescence analysis

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL- NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands.