In vitro activity: BAF312 (Siponimod) is a potent and selective S1P receptor agonist, with EC50 of 0.39 nM and 0.98 nM for S1P1 and S1P5receptors, exhibits >1000-fold selectivity over S1P2, S1P3 and S1P4 receptors. BAF312 (1 h at 1 μM) promotes prominent internalization of S1P1 receptors by 91%.

Kinase Assay: The cells are homogenized and centrifuged at 26900 × g for 30 min at 4°C. Membranes are re-suspended in 20 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA and 0.1% fat-free BSA at 2–3 mg protein/mL. GTPγ[35S] binding assay is performed with the membranes (75 mg protein /mL in 50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 20 μg/mL saponin and 0.1% fat-free BSA (pH 7.4), 5 mg/mL with wheat-germ agglutinin-coated scintillation proximity assay-bead, and 10 μM GDP for 10–15 min. The GTPγ[35S]-binding reaction is started by the addition of 200 pM GTPγ[35S]. After 120 min at room temperature, the plates are centrifuged for 10 min at 300 × g and counted.

Cell Assay: Agonist-mediated internalization of S1P1 receptors in CHO cells analysed by flow cytometry Myc-tag hS1P1 cells are incubated for 1 h with agonist at 37°C in standard culture medium followed by a PBS wash. An aliquot is kept on ice for 3 h, while another aliquot is left for 3 (or 12) h in culture medium (no agonist) at 37°C. The cells are then incubated either with 4 μg/mL monoclonal mouse anti C-myc IgG1 antibody or with isotype control mouse IgG1 for 60 min at 4°C, followed by an incubation with 1 μg/mL of Alexa488-labelled goat anti-mouse secondary conjugates. The cells are subjected to flow cytometry measurements using 10000 viable cells per sample.