CYM-5541 (ML249) is a selective and allosteric S1P3 receptor agonist.

S1P3:72-132 nM(EC50)

CYM-5541 is a full agonist that achieves maximal ERK phosphorylation levels comparable to S1P, with an EC50 of 72-132 nM and high selectivity over other S1P receptor subtypes: S1P1 EC50 >10 μM, S1P2 EC50 >50 μM, S1P4 EC50 >50 μM, and S1P5 EC50 >25 μM. It exhibits no significant activity in the Ricerca profiling panel of 55 GPCRs, ion channels, and transporters. CYM-5541 enabled the identification of an allosteric site, with residue F263 being crucial for its affinity and efficacy, suggesting the presence of a unique hydrophobic pocket responsible for its S1P3 selectivity [1].

Jump-In TI CHO-K cells stably expressing WT or mutant S1P3 are serum-starved for 4 hrs. They are 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 with 0.1 nM [33P]S1P and increasing concentrations of S1P, SPM-242, or CYM-5541. Cells are washed three times with cold binding buffer. Cell-bound radioactivity is measured by lysing the cells with 0.5% SDS followed by liquid scintillation counting. The raw data is normalized so that the level of [33P]S1P bound to each cell line (WT or mutant) in the absence of competing ligand is referenced as 100% for its own cell line[1].