A novel, time resolved immunofluorescence screening assay to assess PAR2 ligand binding

We describe the synthesis and use of a modified PAR 2 peptidomimetic agonist, 2‐furoyl‐LIGRLO‐DTPA‐NH 2 (2‐f‐ DTPA), for lanthanide (Europium)‐based time resolved fluorescence screening. We compared 2‐f‐DTPA to its parent compound, the PAR 2 peptidomimetic 2‐furoyl‐LIGRLO‐NH 2 , across a full spectrum of in vitro assays to demonstrate its potency and specificity as a PAR 2 agonist. 2‐f‐DTPA induced full PAR 2 ‐ specific in vitro physiological responses. Current PAR 2 ligand screening methods utilize radiolabeled ligands and/or have low throughput screening capabilities. The advantage of our 2‐f‐DTPA ligand is that it can be used in a radioactivity‐free, high throughput screen without sacrificing sensitivity. We used 2‐f‐DTPA in a competitive binding assay to evaluate ligand‐receptor interactions at PAR 2 with known high potency agonists (2‐aminothiazol‐4‐yl‐ LIGRL‐NH 2 and; 6‐aminonicotinyl‐LIGRL‐NH 2 ) and a negative control (3‐indolacetyl‐LIGRL‐NH 2 ). Both agonists demonstrated competitive binding reflecting their in vitro potency, whereas 3‐ indolacetyl‐LIGRL‐NH 2 displayed limited competition for PAR 2 binding. In summary, we report a PAR 2 agonist that can be used in a highly sensitive competitive binding assay to screen novel PAR 2 ligands in a high‐throughput manner. Supported by NIH grant NS073664.