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Identification of Leu276 of the S1P₁ Receptor and Phe263 of the S1P₃ Receptor in Interaction with Receptor Specific Agonists by Molecular Modeling, Site-Directed Mutagenesis, and Affinity Studies

Departments of Molecular Systems (Q.D., R.M.), Immunology & Rheumatology (J.A.C., G.C., J.B., S.M. S.A.P.), Cardiovascular Diseases (P.F.), and Medicinal Chemistry (J.J.H., Z.L., S.G.M.), Merck Research Laboratories, Rahway, New Jersey

Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P₁ against S1P₃ is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P₁ and S1P₃ receptors. Computational models of human S1P₁ and S1P₃ receptors bound with two nonselective agonists or two S1P₁-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P₁ Leu276 and S1P₃ Phe263 contribute to the S1P₁/S1P₃ selectivity of the two S1P₁-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P₁-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P₁ and S1P₃ receptors, which should aid development of more selective S1P₁ receptor agonists with immunosuppressive properties and improved safety profiles.

Address correspondence to: Dr. Stephen A. Parent, Merck Research Laboratories, PO Box 2000, RY80Y-225, Rahway, NJ 07065. E-mail: steve_parent{at}merck.com