Abstract

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor, a G protein-coupled receptor that mediates chemotaxis of inflammatory cells in response to prostaglandin D₂ (PGD₂), is hypothesized to play a role in Th2-mediated allergic disease. In addition to PGD₂, CRTH2 can be activated by indomethacin, a nonselective cyclooxygenase inhibitor and widely used nonsteroidal anti-inflammatory drug (NSAID). To evaluate the structural features that confer CRTH2 binding selectivity, structure-activity relationship analysis of arylacetic acid class NSAIDs as CRTH2 receptor ligands was performed. Indomethacin, sulindac sulfide, and zomepirac displaced [³H]PGD₂ binding at the mouse CRTH2 receptor (mCRTH2) with comparable affinity (K_i = 1.5 ± 0.1, 2.5 ± 0.4, and 3.3 ± 0.3 μM, respectively). The indomethacin metabolite 5′-O-desmethyl indomethacin (5′-DMI) possessed binding affinity similar to indomethacin; however, elimination of the 2-methyl substituent on the indole ring resulted in a 10-fold decrease in binding affinity. No binding was detected for indole acetic acid and indole derivatives such as tryptophan, serotonin, and 5-hydroxy indole acetic acid, demonstrating the importance of the N-acyl moiety of indomethacin. Neutral derivatives of indomethacin also failed to bind to mCRTH2, suggesting that the negatively charged carboxylate moiety participates in a key ligand-receptor interaction. Despite similar binding affinities, NSAID-type mCRTH2 ligands exhibited variable potencies as mCRTH2 agonists. Sulindac sulfide and 5′-DMI inhibited intracellular cyclic AMP ([cAMP]_i) generation and stimulated cell migration comparable with indomethacin. In contrast, zomepirac did not inhibit [cAMP]_i generation or stimulate cell migration but weakly antagonized the effects of indomethacin on [cAMP]_i. Together, these results reveal structural features of arylacetic acid NSAIDs that may be exploited for the development of selective CRTH2 ligands.