Mutations in the S1P1 binding pocket differentially affect S1P and FTY720-P-induced signalling

Pieter B. van Loenen1, Chris de Graaf2, Dennis Verzijl1, Rob Leurs2, Didier Rognan3, Stephan L.M. Peters1, Astrid E. Alewijnse1. 1Academic Medical CenterPharmacology and Pharmacotherapy, 1105 AZ Amsterdam, Netherlands, 2VU University, Division of Medicinal Chemistry, 1081 HV Amsterdam, Netherlands, 3Université de Strasbourg, Structural Chemogenomics Group, F-67400 Illkirch, France.

The sphingosine-1-phosphate type 1 (S1P1) receptor belongs to the family of G protein-coupled S1P receptors, a class of five receptors which play important roles in the cardiovascular system and in immune regulation. FTY720 is a novel immunomodulatory drug which recently has been approved by the FDA for the treatment of multiple sclerosis. The phosphorylated form of this compound, FTY720-P (Albert et al., 2005) has affinity for all S1P receptor subtypes except the S1P2 receptor. The S1P1 receptor seems to be to most important target for the immunomodulatory effects observed for FTY720. Compared to S1P, the natural ligand for S1P receptors, FTY720-P, has some unique pharmacological features. Ample evidence indicates that upon S1P stimulation S1P1 receptors internalize and recycle back to the cell membrane whereas FTY720-P induces S1P1 receptor internalization and subsequent receptor degradation. A likely explanation for the differences in pharmacological properties between S1P and FTY720-P is that S1P and FTY720-P stabilize different S1P1 conformations. In this study we investigate the differences in ligand-induced receptor activation between S1P and FTY720-P using an in silico guided site-directed mutagenesis strategy. The S1P1 WT, as well as the mutant receptors, where stably expressed in Chinese Hamster Ovary cells and signal transduction was measured by determining the effect of the ligands on the forskolin-induced cAMP accumulation.

This study for the first time shows that, in contrast to other S1P1 modelling studies but in line with our receptor modelling studies, Y2.57 has no direct role in S1P1 receptor-ligand interactions. Removing the hydroxyl group of the Y2.57 residue (Y2.57F) does not affect S1P or FTY720-P-induced signalling. However, replacing the Y2.57 residue by an alanine residue (Y2.57A) decreased the potency of S1P (pEC50 9.8 ± 0.1 versus 7.9 ± 0.1) whereas the potency of FTY720-P is not affected (pEC50 10.4 ± 0.1 versus 10.5 ± 0.1). Based on these data and our modelling studies we conclude that the Y2.57 residue is indirectly involved in the positioning of R3.28, a residue known to be crucial in S1P1 receptor-ligand interactions. The insensitivity of FTY720-P to the Y2.57A mutation is most likely explained by the fact that, compared to other ligands, the polar head of FTY720-P is relatively flexible and can more easily adapt to changes in the binding pocket. In addition this study shows that, compared to non-aromatic ligands, the binding mode of FTY720-P is additionally stabilized by aromatic stacking with F3.33, as confirmed by site-directed mutagenesis of this ligand binding residue in S1P1.

In conclusion, our data support the hypothesis that FTY720-P is able to stabilize specific active receptor conformations that cannot be stabilized by S1P and gives new insights into the molecular basis for the unique pharmacological properties of FTY720-P.

Albert et al. (2005) J. Med. Chem. 48:5373-5377.