In vitro activity: CYM-5541 (also known as ML249) is a potent, selective, and allosteric agonist of the S1P3 (Sphingosine 1-phosphate) receptor with EC50 in the range of 72 to 132 nM. S1P is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating G protein-coupled receptors for S1P, namely, S1P(1) through S1P(5). CYM-5541 was used to to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety, and the novel allosteric hydrophobic pocket permits S1P(3) selectivity of CYM-5541 within the highly similar S1P receptor family.

Kinase Assay:

ELISA for p44/42 MAPK Phosphorylation: Ligand-mediated ERK phosphorylation was measured using PathScan Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA kit (Cell Signaling Technology). Cells expressing WT or mutant S1P3were serum-starved for 4 hr. In the antagonist experiments, cells were either pre-incubated with SPM-242 at 1 uM for 15 min before agonist treatment or 1 uM SPM-242 was pre-mixed with agonists. Cells were then stimulated for 5 min (determined to give maximal ERK phosphorylation for all agonists) with increasing concentrations of S1P or CYM-5541 and phosphorylation of p44/42 MAPK was assayed according to manufacturer’s protocol. The dose response curves for agonist-mediated p44/42 MAPK phosphorylation were analyzed and EC50 was determined using Prism (Graphpad Software).

[33P]S1P Radioligand Binding Assay: Jump-In TI CHO-K cells (5 × 105) stably expressing WT or mutant S1P3 were serum-starved for 4 hrs. They were then incubated at 4 °C for 30 min in the binding buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na3VO4, 0.5% fatty acid-free bovine serum albumin, and protease inhibitor mixture (Roche) with 0.1 nM [33P]S1P and increasing concentrations of S1P, SPM-242, or CYM-5541. Cells were washed three times with cold binding buffer. Cell-bound radioactivity was measured by lysing the cells with 0.5% SDS followed by liquid scintillation counting. The raw data was normalized so that the level of [33P]S1P bound to each cell line (WT or mutant) in the absence of competing ligand was referenced as 100% for its own cell line.

Cell Assay: Plasmid encoding N-terminal, triple HA-tagged S1P3 fusion protein in pcDNA3.1 was purchased from cDNA.org. S1P3 receptor mutants were generated by overlapping PCR mutagenesis, and sequences were verified prior to use. Jump-In(TM) TI(TM) CHO-K parental cells, Gateway cloning vectors (pDONR 221, pJTI R4 DEST, and pJTI R4 Int), and enzymes (BP clonase II and LR clonase) were purchased from Invitrogen Corp. Triple HA-tagged WT and mutant S1P3 were first cloned into entry clones using the BP recombination reaction. They were retargeted into an appropriate pJTI R4 DEST vector to yield a pJTI R4 EXP retargeting expression vector. Jump-In TI CHO-K cells were transfected with both pJTI R4 EXP S1P1 and pJTI R4 Int vector for retargeting. After expanding retargeted cells, retargeted Jump-In TI cells were selected using blasticidin at 10 μg/mL for 4 weeks.