S1PR2 (sphingosine 1-phosphate receptor 2)
CYM-5520 is a selective allosteric agonist of the Sphingosine 1-phosphate receptor 2 (S1PR2). Its EC₅₀ for activating S1PR2 is reported as 0.48 µM in a CRE-bla reporter assay and 1.6 µM in a cAMP biosensor assay using wild-type S1PR2-expressing cells. [1]
It does not activate S1PR1, S1PR3, S1PR4, or S1PR5 at concentrations up to 10 µM. [1]

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].

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CYM-5520 activates S1PR2 in a concentration-dependent manner, acting as a full agonist in both CRE-bla reporter gene assays and intracellular cAMP measurement assays. [1]
It is highly selective for S1PR2, showing no significant activity (>20% inhibition) in a panel of 29 other receptors and transporters (PanLabs HitProfiling Screen). [1]
The compound acts as an allosteric agonist, as evidenced by its inability to displace radiolabeled S1P ([³³P]-S1P) from S1PR2 in competitive binding assays, and by its retained agonist activity on an S1PR2 triple mutant (R108A, E109A, K269A) receptor, which is unresponsive to the native ligand S1P. [1]
In the presence of the orthosteric antagonist JTE-013, the dose-response curve of CYM-5520 is shifted to the right and the maximal response is diminished, indicative of a non-competitive interaction. [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).

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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.

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S1PR2 CRE-bla Reporter Assay: Stable CHO cells expressing S1PR2 and a CRE-driven β-lactamase reporter gene were used. Cells were treated with serial dilutions of CYM-5520 or control agonists. After an incubation period (duration not specified in detail), β-lactamase activity was measured via a fluorescent substrate readout (blue/green ratio) to determine receptor activation. EC₅₀ values were calculated from dose-response curves. [1]
cAMP Biosensor Assay (GloSensor): CHO cells stably expressing either wild-type or triple mutant (R108A, E109A, K269A) S1PR2-eGFP were transiently transfected with a cAMP-sensitive luciferase biosensor vector. Cells were plated, loaded with the luciferase substrate, and treated with CYM-5520 or S1P. Luminescence was measured after 15 minutes to quantify intracellular cAMP levels as an indicator of receptor activation (S1PR2 couples to Gs). [1]
Radioligand Competition Binding Assay: S1PR2-expressing cells were incubated with a fixed concentration of [³³P]-S1P and increasing concentrations of unlabeled S1P, JTE-013, or CYM-5520 in binding buffer at 4°C. Cells were then processed to measure bound radioactivity. The inability of CYM-5520 to displace [³³P]-S1P was demonstrated. [1]
Antagonist Inhibition Assay: In the S1PR2 CRE-bla reporter assay, dose-response curves for CYM-5520 were generated in the presence of increasing fixed concentrations of the antagonist JTE-013 to assess the nature of the interaction (competitive vs. non-competitive). [1]

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.

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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 compounds targeting sphingosine-1-phosphate receptor 2 (S1PR2) are crucial for studying various biological processes involved in the S1PR2 receptor, including NF-κB-mediated tumor cell survival and fibroblast chemotaxis of fibronectin. This paper reports our screening and characterization of S1PR2-selective chemical probes. Using a high-throughput screening method, we identified two compounds capable of activating the S1PR2 receptor. Structure-activity relationship optimization yielded compounds with nanomolar-level high activity. These compounds, XAX-162 and CYM-5520, exhibit high selectivity and do not activate other S1P receptors. CYM-5520 binds non-competitively to the antagonist JTE-013. Mutations in receptor residues (responsible for binding to the zwitterionic head group of sphingosine-1-phosphate (S1P)) eliminate S1P activation of the receptor but do not eliminate the activation of CYM-5520. Competitive binding experiments with radiolabeled S1P showed that CYM-5520 is an allosteric agonist and does not replace the natural ligand. Computational models showed that CYM-5520 binds to the lower position of the ortho-binding pocket and co-binding with S1P is energy-tolerant. In summary, we identified an S1PR2 selective allosteric agonist compound. [1]

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Molecular probe tools for sphingosine-1-phosphate receptor 2 (S1PR2) are crucial for studying a variety of biological processes involving the S1PR2 receptor. These include NF-κB-mediated tumor cell survival and fibroblast chemotaxis of fibronectin. This paper reports our work on identifying and characterizing S1PR2 selective chemical probes. We used a high-throughput screening method to identify two compounds that can activate the S1PR2 receptor. Structure-activity relationship optimization (SAR) ultimately yielded compounds with nanomolar high activity. These compounds, XAX-162 and CYM-5520, exhibit high selectivity and do not activate other S1P receptors. CYM-5520 binds non-competitively to the antagonist JTE-013. Mutations in the receptor residues responsible for binding to the zwitterionic head group of sphingosine-1-phosphate (S1P) eliminate S1P activation but not CYM-5520 activation. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist that does not replace the natural ligand. Computational models indicate that CYM-5520 binds to a lower position in the ortho-binding pocket and exhibits good energy tolerance to co-binding with S1P. In summary, we have identified an allosteric S1PR2 selective agonist compound. [2]

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CYM-5520 was obtained from the initial lead compound (CYM-5482) through high-throughput screening (HTS) and subsequent structure-activity relationship (SAR) optimization. [1]
It is a synthetic small molecule lacking the zwitterionic head group of the natural ligand S1P. Computational modeling and molecular docking studies suggest that it binds to the hydrophobic region below the ortho-binding pocket of S1PR2 and may interact with residue F274, which may contribute to its subtype selectivity. The model also suggests that CYM-5520 can co-bind with S1P in the acceptor pocket. [1]

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.

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