CYM5520

S1PR2 (sphingosine 1-phosphate receptor 2)

CYM-5520 是野生型 S1PR2 的完全激动剂，EC50 为 1.6 μM。虽然 CYM-5520 是一种 EC50 为 1.5 μM 的激动剂，但用 S1P 刺激表达三重突变体 S1PR2 的细胞不会刺激荧光素酶活性 [1]。

骨质减少切除手术后，CYM-5520（10 mg/kg；腹腔注射；每周 5 天；持续 6 周）治疗显着增加长骨和椎骨骨量。此外，CYM-5520 还可提高碱性磷酸酶、成骨细胞计数和前胶原 IC 末端前肽（骨合成代谢标志物）的浓缩浓度 [2]。

33P-S1P放射性配体竞争结合试验[1]
采用Sphingosine， D-erythro [33P] 1-phosphate。将S1PR2-CRE bla细胞接种到24孔板的孔中，20万个细胞加入1.0 mL生长培养基，在100%湿度、5% co2、37℃的培养箱中培养过夜。检测前将培养基替换为1% CDS血清培养基4小时。在4°C时，将培养基去除，并在结合缓冲液（20 mM Tris, pH 7.5, 100 mM NaCl， 15 mM NaF和新鲜添加的1 mM Na3VO4和蛋白酶抑制剂）中替换为测试化合物或载体对照。

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Glo-sensor cAMP瞬时转染试验[1]
使用Fugene HD将GloSensor载体（pGLoSensor-20FcAMP, Promega）转染到S1PR2-eGFP或S1PR2-TM-eGFP Jump-In CHO细胞中。第二天，用0.05%胰蛋白酶EDTA收获细胞，在含有2%糖葡聚糖剥离血清的CO2独立培养基中重悬至500,000个细胞/mL，加入20 μL细胞悬液于384孔组织培养处理过的白板中，在37℃，5% CO2条件下孵育过夜。然后加入25 uL含有2%CDS和4% GloSensor Reagent的CO2独立培养基，室温孵育2小时。加入拮抗剂（JTE-013）或对照物孵育20分钟，然后加入激动剂化合物或S1P。15分钟后，在Perkin Elmer Envision平板阅读器上读取发光。

Jump-In CHO S1PR2野生型和头组三突变细胞系[1]
利用多位点Gateway克隆技术，从pEnter-15- S1pr2和pENTER-52-eGFP中获得帧内S1pr2- egfp表达构建体。将S1pr2-eGFP融合蛋白表达载体克隆到pDEST-CMV-JTI中。通过与S1PR1比对，确定了S1PR2头基团结合侧链。这些突变是通过重叠寡核苷酸PCR产生的。14通过重叠寡核苷酸PCR诱变产生三突变体S1PR2 （R108A、E109A和K269A）。所有构建体均经DNA测序证实。将这些载体转染到CHO JumpIn细胞中，并用10微克/毫升囊胚杀虫素进行筛选通过流式细胞仪对GFP阳性细胞进行分选，形成了一个均匀的细胞池。

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 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]

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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

66.1

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

注意： 本产品在运输和储存过程中需避光。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.24 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。