S1PR2 (sphingosine 1-phosphate receptor 2)

CYM-5520 is a complete agonist of wild-type S1PR2 with an EC50 of 1.6 μM. While CYM-5520 is an agonist with an EC50 of 1.5 μM, stimulating cells expressing the triple mutant S1PR2 with S1P does not stimulate luciferase activity [1].

Following osteopenia resection surgery, CYM-5520 (10 mg/kg; intraperitoneal injection; 5 days per week; for 6 weeks) treatment significantly increased long bone and vertebral bone mass. In addition, CYM-5520 raises alkaline phosphatase, osteoblast counts, and the concentrated concentration of procollagen IC-terminal propeptide, a marker of bone anabolism [2].

33P-S1P Radioligand Competition Binding Assay[1]
Sphingosine, D-erythro [33P] 1-phosphate was used. S1PR2-CRE bla cells were seeded into wells of a 24 well plate at 200,000 cells in 1.0 mL growth media and the plate incubated overnight in an incubator with 100% humidity, 5% C02, 37°C. The media was replaced with 1% CDS serum media for 4 hours prior to the assay. At 4°C, the media was removed and replaced with test compounds or vehicle controls in binding buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 15 mM NaF with freshly added 1 mM Na3VO4 and protease inhibitors).

---

Glo-sensor cAMP transient transfection assay[1]
The GloSensor vector (pGLoSensor-20FcAMP, Promega) was transfected using Fugene HD into S1PR2-eGFP or S1PR2-TM-eGFP Jump-In CHO cells. The following day, cells were harvested with 0.05% trypsin EDTA, resuspended to 500,000 cells/mL in CO2 independent Media containing 2% charcol dextran stripped serum and 20 μL of the cell suspension was added to 384 well tissue culture treated white plates These plates were incubated overnight at 37 °C, 5% CO2. 25 uL of CO2 independent media containing 2%CDS and 4% GloSensor Reagent were then added and the plates were incubated for 2 hours at room temperature. Antagonist (JTE-013) or vehicle were added and incubated for 20 minutes followed by agonist compounds or S1P. After 15 minutes, luminescence was read on a Perkin Elmer Envision plate reader.

Jump-In CHO S1PR2 wild type and head group triple mutant cell lines [1]
Multisite Gateway cloning was utilized to generate in-frame S1pr2-eGFP expression constructs from pEnter-15- S1pr2 and pENTER-52-eGFP. The S1pr2-eGFP fusion protein expression vector was cloned into pDEST-CMV-JTI. S1PR2 head group binding side chains were identified by alignment with S1PR1. These mutations were generated by overlapping oligonucleotide PCR.14 The triple mutant S1PR2 (R108A, E109A and K269A) was generated by overlapping oligonucleotide PCR mutagenesis. All constructs were confirmed by DNA sequencing. These vectors were transfected into CHO JumpIn cells and selected with 10 microgram/mL blasticidin as described.15 A homogenous pool of cells was generated by FACS sorting of GFP positive cells.

Animal/Disease Models: Ovariectomized 12weeks old C57Bl6J mice[2]
Doses: 10 mg/kg
Route of Administration: ip; 5 days per week; 6 weeks
Experimental Results: Correction of ovariectomized mice by inducing new bone formation Osteopenia.

---

Ovariectomized 12 weeks old C57Bl6J mice were purchased from Charles River Laboratories. Mice were housed in SPF cages without any pathogens and with access to mouse chow and water ad libitum. Treatment was started 5 weeks after OVX. 1E)-1-(4-((1R,2S,3R)-1,2,3,4-Tetrahydroxybutyl)-1H-imidazol-2-yl)ethanone oxime (LX2931) was synthesized according to the known procedure and administered with the drinking water at 200 mg/kg/day for 6 weeks. CYM5520 was administered intraperitoneally at 10 mg/kg/day for 5 consecutive days per week for 6 weeks. The total number of mice used in the whole study was 21. Every effort was taken to minimize the number of animals used and their suffering.[2]

[1]. A sphingosine 1-phosphate receptor 2 selective allosteric agonist. Bioorg Med Chem. 2013 Sep 1;21(17):5373-82.

[2]. Agonist-induced activation of the S1P receptor 2 constitutes a novel osteoanabolic therapy for the treatment of osteoporosis in mice. Bone. 2019 Aug;125:1-7.

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. [1]

---

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.[2]

C21H19N3O2

345.394464731216

345.147

C, 73.03; H, 5.54; N, 12.17; O, 9.26

1449747-00-5

25110470

White to off-white solid powder

1.2±0.1 g/cm3

580.5±50.0 °C at 760 mmHg

304.9±30.1 °C

0.0±1.6 mmHg at 25°C

1.611

2.52

0

3

5

26

666

0

FMYGNANMYYHBSU-UHFFFAOYSA-N

InChI=1S/C21H19N3O2/c1-15-10-19(16(2)24(15)13-17-6-4-3-5-7-17)20(25)14-23-12-18(11-22)8-9-21(23)26/h3-10,12H,13-14H2,1-2H3

1-[2-[2,5-Dimethyl-1-(phenylmethyl)-1H-pyrrol-3-yl]-2-oxoethyl]-1,6-dihydro-6-oxo-3-pyridinecarbonitrile

CYM-5520; CYM5520; 1449747-00-5; 1-[2-(1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl)-2-oxo-ethyl]-6-oxo-1,6-dihydro-pyridine-3-carbonitrile; 1-[2-[2,5-Dimethyl-1-(phenylmethyl)-1H-pyrrol-3-yl]-2-oxoethyl]-1,6-dihydro-6-oxo-3-pyridinecarbonitrile; 1-(2-(1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl)-2-oxoethyl)-6-oxo-1,6-dihydropyridine-3-carbonitrile; 1-[2-(1-BENZYL-2,5-DIMETHYLPYRROL-3-YL)-2-OXOETHYL]-6-OXOPYRIDINE-3-CARBONITRILE; CYM 5520.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~50 mg/mL (~144.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.24 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.

---

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