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

S1P3:72-132 nM(EC50)

CYM-5541是一种全效激动剂，能够达到S1P观察到的ERK磷酸化的最大水平。CYM-5541的EC50介于72和132 nM之间，并且展现出对其他S1P受体亚型的极高选择性：S1P1 EC50>10 μM，S1P2 EC50>50 μM，S1P4 EC50>50 μM，以及S1P5 EC50>25 μM。CYM-5541还在对55种GPCRs、离子通道和转运蛋白的Ricerca分析板中表现出对一大组蛋白靶点的选择性，没有显著的活动。CYM-5541使我们能够识别一个异构位点，在该位点中F263是其亲和力和效力的关键守门氨基酸。这个新颖的异构疏水口袋可能解释了CYM-5541对S1P3的选择性[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].