CXCR 3 antagonist VUF 10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK ‐779

Background and Purpose The chemokine receptor CXCR 3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR 3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR 3 antagonist VUF 10085 and the broad spectrum antagonist TAK ‐779 which blocks CXCR 3 along with CCR 2 and CCR 5. Experimental Approach Molecular modelling of CXCR 3, followed by virtual ligand docking, highlighted several CXCR 3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 ( A sp‐112 2:63 ), which was postulated to interact with the quaternary nitrogen of TAK ‐779. Validation of modelling was carried out by site‐directed mutagenesis of CXCR 3, followed by assays of cell surface expression, ligand binding and receptor activation. Key Results Mutation of A sn‐132 3.33 , Phe‐207 and T yr‐271 6.51 within CXCR 3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR 3 conformation. Contrary to our hypothesis, mutation of A sp‐112 2:63 had no observable effects on TAK ‐779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of P he‐131 3.32 , I le‐279 6.59 and T yr‐308 7.43 were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK ‐779. Conclusions and Implications This more detailed definition of a binding pocket within CXCR 3 for low MW antagonists should facilitate the rational design of newer CXCR 3 antagonists, with obvious clinical potential.