S1P₃ ( EC50: between 72 and 132 nM )
The target of CYM-5541 (ML249) is the sphingosine 1-phosphate receptor 3 (S1P₃), a G protein-coupled receptor (GPCR). It acts as a selective allosteric agonist of S1P₃, [1]

In vitro activity: CYM-5541 is a complete agonist, able to phosphorylate ERK to the highest extent seen with S1P. CYM-5541 demonstrates remarkable selectivity over other S1P receptor subtypes, including S1P1 EC50>10 μM, S1P2 EC50>50 μM, S1P4 EC50>50 μM, and S1P5 EC50>25 μM, with an EC50 ranging from 72 to 132 nM. In the Ricerca profiling panel of 55 GPCRs, ion channels, and transporters, CYM-5541 likewise demonstrates selectivity over a broad panel of protein targets with no discernible activities. Our identification of an allosteric site where F263 is a crucial gate-keeper residue for its efficacy and affinity was made possible by CYM-5541. CYM-5541's S1P3 selectivity could be explained by its unique allosteric hydrophobic pocket[1].

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1. CYM-5541 (ML249) was identified as a full agonist of S1P₃ in WT S1P₃ Jump-In stable CHO-K cell lines; ligand-induced ERK phosphorylation assays showed that it activated S1P₃ in a concentration-dependent manner, with a stimulatory effect comparable to the orthosteric agonist S1P (Mean ± SEM; n=3) [1]
2. Radioligand binding competition assays revealed that CYM-5541 (ML249) was unable to compete with 0.1 nM [³³P]S1P for binding to WT S1P₃, W256L, or F263L mutant S1P₃ receptors, indicating it binds to an allosteric site distinct from the orthosteric pocket of S1P (Mean ± SEM; n=3) [1]
3. Mutagenesis studies demonstrated that the F263L mutation in S1P₃ significantly shifted the receptor activation induced by CYM-5541 (ML249) (ERK phosphorylation assay), while the W256L mutation had no effect on its activity; this confirmed Phe263 as a key gate-keeper residue for the affinity and efficacy of CYM-5541 (ML249) (Mean ± SEM; n=3) [1]
4. Calcium response assays in S1P₃-CHO cells showed that co-application of CYM-5541 (ML249) and S1P led to an additive increase in calcium release, suggesting the two ligands act on distinct binding sites and their effects on S1P₃ activation are synergistic (Mean ± SEM; n=9) [1]
5. CYM-5541 (ML249) exhibited high selectivity for S1P [1]

For four hours, WT or mutant S1P3-expressing Jump-In TI CHO-K cells are serum-starved. Following this, they are incubated for 30 minutes at 4 °C in the binding buffer, which contains 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 15 mM NaF, 0.5 mM EDTA, 1 mM Na₂VO₄, 0.5% fatty acid-free bovine serum albumin, and a protease inhibitor mixture containing 0.1 nM [³³P]S1P and increasing concentrations of S1P, SPM-242, or CYM-5541. Cold binding buffer is used to wash the cells three times. Liquid scintillation counting is the method used to measure cell-bound radioactivity after the cells have been lysed with 0.5% SDS. In order to reference the level of [³³P]S1P bound to each cell line (WT or mutant) in the absence of a competing ligand as 100% for its own cell line, the raw data is normalized[1].

The pcDNA3.1 plasmid encoding the triple HA-tagged N-terminal S1P3 fusion protein was acquired from cDNA.org. Overlapping PCR mutagenesis was used to create S1P3 receptor mutants, and sequences were checked before use. The Gateway cloning vectors (pDONR 221, pJTI R4 DEST, and pJTI R4 Int), enzymes (BP clonase II and LR clonase), and Jump-InTM TI™ CHO-K parental cells were acquired from Invitrogen Corp. The BP recombination reaction was first used to clone triple HA-tagged WT and mutant S1P3 into entry clones. In order to create a pJTI R4 EXP retargeting expression vector, they were retargeted into a suitable pJTI R4 DEST vector. For retargeting, both the pJTI R4 EXP S1P1 and the pJTI R4 Int vector were transfected into Jump-In TI CHO-K cells. Retargeted Jump-In TI cells were selected using blasticidin at 10 μg/mL for four weeks after the cells had expanded.

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1. ERK Phosphorylation Assay in S1P₃ Stable Cell Lines: Targetable Jump-In™ CHO-K cell lines were engineered to integrate a single copy of WT S1P₃ (or mutant variants F263L/W256L) in a site-specific manner. The stable cell lines were cultured under standard conditions and stimulated with increasing concentrations of CYM-5541 (ML249) or S1P. After stimulation, cell lysates were prepared, and ERK phosphorylation levels were detected by Western blotting (or a quantitative phosphorylation assay) to measure S1P₃ activation; data were presented as mean ± SEM of three independent experiments [1]
2. Radioligand Binding Competition Assay: WT, W256L, and F263L S1P₃ Jump-In stable cell lines were incubated with 0.1 nM [³³P]S1P in the presence of escalating concentrations of CYM-5541 (ML249), S1P, or SPM-242. Following incubation, unbound radioligand was removed by washing, and the radioactivity associated with the cells was quantified to determine the competitive binding capacity of each ligand; results were expressed as mean ± SEM of three replicates [1]
3. Calcium Response Assay in S1P₃-CHO Cells: S1P₃-CHO cells were seeded in suitable plates and loaded with a calcium-sensitive fluorescent dye. CYM-5541 (ML249) was applied alone or in combination with S1P, and the fluorescence intensity was monitored in real-time to measure calcium release; the assay was repeated nine times, with data presented as mean ± SEM [1]
4. NFAT β-Lactamase Reporter Assay: S1P₃-expressing reporter cell lines were treated with CYM-5541 (ML249) at different concentrations (10⁻⁹·⁵ to 10⁻⁷ M) in the presence or absence of SPM-242. The β-lactamase activity (a readout of NFAT signaling downstream of S1P₃) was measured using a fluorescent substrate, and the dose-response curve shift was analyzed to evaluate the antagonistic effect of SPM-242 on CYM-5541 (ML249); data were shown as mean ± SEM of three replicates [1]

[1]. Novel selective allosteric and bitopic ligands for the S1P(3) receptor. ACS Chem Biol. 2012 Dec 21;7(12):1975-83.

1. CYM-5541 (ML249) is a novel selective S1P₃ allosteric agonist with the chemical structure N,N-dicyclohexyl(5-cyclopropylisoxazol-3-yl)formamide; it lacks a polar moiety and binds to the hydrophobic allosteric pocket of S1P₃, which endows it with subtype selectivity in the S1P receptor family [1]
2. The binding mode of CYM-5541 (ML249) to S1P₃ was elucidated by combining site-directed mutagenesis, ligand competition experiments and molecular modeling (homology modeling based on S1P₁ crystal structure and molecular dynamics simulation); compared with the ortho-agonist S1P, this ligand occupies a different chemical space in the S1P₃ binding pocket [1]
3. CYM-5541 (ML249) is a valuable tool compound for studying the allosteric regulation of S1P₃ and its physiological functions [1]

C19H28N2O2

316.45

316.215

C, 72.12; H, 8.92; N, 8.85; O, 10.11

945128-26-7

17253208

White to off-white solid powder

1.1±0.1 g/cm3

503.1±38.0 °C at 760 mmHg

258.1±26.8 °C

0.0±1.3 mmHg at 25°C

1.559

3.47

0

3

4

23

394

0

O=C(N(C1CCCCC1)C1CCCCC1)C1C=C(C2CC2)ON=1

NDKGACIWVAOUQH-UHFFFAOYSA-N

InChI=1S/C19H28N2O2/c22-19(17-13-18(23-20-17)14-11-12-14)21(15-7-3-1-4-8-15)16-9-5-2-6-10-16/h13-16H,1-12H2

N,N-dicyclohexyl-5-cyclopropyl-1,2-oxazole-3-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)