Agonist-dependent effects of mutations in the sphingosine-1-phosphate type 1 receptor

The sphingosine-1-phosphate type 1 (S1P1) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P1 receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P1 receptor activation using an in silico guided site-directed mutagenesis strategy. S1P1 mutant receptors (modifications of residues Y982.57, R1203.28, F1253.33) were probed with a chemically diverse set of S1P1 agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R3.28 residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y2.57F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y2.57A mutant. The F3.33A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P1 receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R3.28 and 2) the ability of the agonist to make aromatic π-stacking interactions with F3.33. Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y2.57A mutant.