CDN-1163

SERCA (sarco/endoplasmic reticulum Ca2+-ATPase)

用 CDN1163（10 μM；24 小时；大鼠心肌细胞）治疗可降低高血糖产生的核 NFATc 和抵抗素表达，并以时间依赖性方式增强 AMPKα 磷酸化 [2]。
CDN1163显著消除葡萄糖刺激的NFATc核易位。同样，当CDN1163存在时，高糖诱导的抵抗素和细胞核NFATc的表达显著降低，而AMPKα的磷酸化以时间依赖性的方式增加，这表明CDN1163介导的Serca2a功能激活以类似于Serca2a基因转移的方式影响抵抗素和NFATc的表达模式。有趣的是，CDN1163还降低了nfatc介导的抵抗素启动子荧光素酶活性[2]。 CDN1163提高SERCA2 Ca2+- atp酶活性，降低内质网应激诱导的体外细胞死亡。[1]

雄性 ob/ob 和瘦 ob/+ 小鼠腹膜内 (ip) 给予 50 mg/kg CDN1163，持续五天。这种治疗改善了肝脏 Ca2+ 转运活性，减少了体外内质网 (ER) 应激诱导的细胞死亡，并增加了 SERCA2 Ca2+-ATPase 活性。在 ob/ob 小鼠中，CDN1163 降低血糖水平，增强代谢参数和糖异生基因表达，逆转肝脂肪变性，防止 ER 应激和 ER 应激诱导的细胞凋亡，并提高线粒体效率 [1]。

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按上述方法用CDN1163处理Ob/ Ob小鼠4周，用免疫印迹法分析心脏NFATc和抵抗素水平。事实上，与载药处理小鼠相比，cdn1163处理小鼠的心脏显示抵抗素(图8F,G)和核NFATc(图8F,H)蛋白表达显著降低。与抵抗素表达的下降一致，与载药处理相比，CDN1163处理增加了ob/ob小鼠心脏中的AMPKα活性/磷酸化(图8F, 1)。总之，这些结果表明CDN1163能够在体外和体内调节抵抗素的表达，验证了Serca2a的药理激活作为高抵抗性疾病的治疗方法。[2]

利用荧光共振能量转移(FRET)，我们在重组膜系统中进行了高通量筛选(HTS)，寻找能够逆转sarco-/内源性内质网ca - atp酶(SERCA)被其内源性调节剂磷蛋白(PLB)抑制的化合物。长期以来，人们一直在寻找这种化合物来纠正心力衰竭中Ca2+的异常调节。在有或没有受体- plb的磷脂膜中重建供体- serca，并在稳态荧光微孔板读取器中测量FRET。一个包含20000个化合物的文库被重复测试。降低FRET超过三个标准差的化合物被认为是成功的。在43个主要命中(0.2%)中，经过更彻底的检测，发现31个(72%)为假阳性。剩下的12个靶点在ca - atp酶活性测定中进行了测试，其中6个靶点显著激活了SERCA，激活率高达60%，有几个靶点还增强了心肌细胞的收缩性。这些化合物直接激活来自心脏和其他组织的SERCA。这些结果验证了我们的FRET方法，并为药物化学和临床前测试奠定了基础。我们担心由于稳态荧光的低精度而导致的高假阳性率。初步研究表明，新型荧光寿命板阅读器的精度提高了20倍。这种仪器可以显著提高未来高温成像的质量。[3]

蛋白质印迹分析 [2]
细胞类型：大鼠心肌细胞 (H9c2)
测试浓度： 10 μM
孵育时间：24小时
实验结果：高糖诱导的抵抗素和核NFATc表达显着减少。 AMPKα 的磷酸化以时间依赖性方式增加。

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应激细胞活力测定[1]
细胞在96孔板(n = 6)中生长，与未处理的对照组相比，暴露于CDN1163 (10 μm)或DMSO中2 h，然后添加200 μm H2O2。H2O2激活未折叠蛋白反应，是内质网应激促进细胞凋亡的诱导剂。孵育16 h后，使用CellTiter-Glo®Luminescent cell viability Assay测定细胞活力[1]。

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大鼠心肌细胞(H9c2)在添加10%牛血清和1X笔/链球菌抗生素混合物的DMEM中生长。Cells were either infected with Ad. βgal, Ad. Serca2a (at different multiplicity of infection as indicated) and Ad. NFATc or were exposed to CDN1163 (10 μM)， 1,2- bis(2-氨基苯氧基)乙烷-N,N,N '，N ' -四乙酸四基(乙酰氧基甲酯)/BAPTA-AM (2 μM)，或Ca2+ (4 mM)低糖(5 mM)或高糖(25 mM)中模拟糖尿病情况指定时间(添加20 mM甘露醇以验证葡萄糖诱导的渗透作用)。细胞被裂解并收获用于实时pcr和western分析。[2]

Animal/Disease Models: Male 8-10 week old ob/ob mice and lean ob/+ mice [1]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; continued for 5 days
Experimental Results: Dramatically diminished fasting blood glucose and improved Glucose tolerance, improves hepatic steatosis but does not change blood glucose levels or body weight. The expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue increases, and the hepatic expression of genes involved in gluconeogenesis and lipogenesis is diminished, attenuating the ER stress response and ER stress-induced apoptosis, and may Improved mitochondrial biogenesis through SERCA2-mediated activation of the AMP-activated protein kinase pathway.

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Animals Male 8–10-week old ob/ob mice (n = 20) and lean ob/+ mice (n = 10) were used. Obese and lean mice were divided into four groups and treated with either vehicle (10% DMSO, 10% Tween 80 in 0.9% NaCl) or CDN1163 (50 mg/kg) intraperitoneally for 5 consecutive days (day 0 to day 4).[1]

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Glucose and Insulin Tolerance Tests [1]
Ten-hour fasting blood glucose levels were measured in whole blood drawn from the tail vein using the OneTouch Ultra 2 Meter. Both the glucose and insulin tolerance tests were performed after a 10-h fast and an additional 2-h CDN1163 injection, with baseline blood glucose measurement taken before the start of the test. For the glucose tolerance testing, d-glucose dissolved in 0.9% NaCl was delivered intraperitoneally at a dose of 1 g/kg. For the insulin tolerance testing, insulin was administered intraperitoneally at a dose of 1 IU/kg. Blood glucose was measured at the indicated time points.[1]

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Clinical Chemistry[1]
A separate cohort of C57BL/6 mice (10 weeks old, n = 10–20) were either untreated or treated with vehicle or 50 mg/kg CDN1163 as indicated above for a total of 6 weeks. Animals were euthanized, and terminal blood samples were collected (∼0.5 ml); sera were analyzed for a panel of clinical chemistry parameters.[1]

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Male 8 to 10-week old ob/ob mice (B6.Cg-Lepob/J; 000632) and lean ob/+mice (C57BL/6J; 000664) were obtained from Jackson Laboratory (n = 10/group). Mice were divided into 6 groups: lean and ob/ob treated with either vehicle (10% DMSO, 10% Tween 80 in 0.9% NaCl) or CDN1163 (50 mg/kg), intraperitoneally 3×/week for 30 days (pharmacology protocol); lean and ob/ob injected with AAV9.Empty or AAV9.Serca2a (3 × 1012 viral particles) via tail-vein for 12 weeks (gene therapy protocol). Male 8–10-week old Serca2a−/− mice (n = 10) were generated as described previously[2].

[1]. Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders. J Biol Chem. 2016 Mar 4;291(10):5185-98.

[2]. A role for calcium in resistin transcriptional activation in diabetic hearts. Sci Rep. 2018 Oct 23;8(1):15633.

CDN1163 is a secondary carboxamide resulting from the formal condensation of the carboxy group of 4-isopropoxybenzoic acid with the primary amino group of 2-methylquinolin-8-amine. An allosteric activator of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA). It has a role as a SERCA activator. It is a member of quinolines, a secondary carboxamide and an aromatic ether.

C20H20N2O2

320.39

320.152

C, 74.98; H, 6.29; N, 8.74; O, 9.99

892711-75-0

892711-75-0

16016585

White to off-white solid powder

1.2±0.1 g/cm3

430.4±35.0 °C at 760 mmHg

214.1±25.9 °C

0.0±1.0 mmHg at 25°C

1.644

5.08

51.2Ų

1

3

4

24

418

0

GVGVYDCVFBGALZ-UHFFFAOYSA-N

InChI=1S/C20H20N2O2/c1-13(2)24-17-11-9-16(10-12-17)20(23)22-18-6-4-5-15-8-7-14(3)21-19(15)18/h4-13H,1-3H3,(H,22,23)

4-(1-Methylethoxy)-N-(2-methyl-8-quinolinyl)benzamide

CDN 1163; CDN1163; 4-isopropoxy-N-(2-methylquinolin-8-yl)benzamide; CDN-1163; N-(2-methylquinolin-8-yl)-4-propan-2-yloxybenzamide; 4-(1-Methylethoxy)-N-(2-methyl-8-quinolinyl)benzamide; N-(2-methylquinolin-8-yl)-4-(propan-2-yloxy)benzamide; CDN 1163; CDN-1163

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)

DMSO : ~100 mg/mL (~312.12 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.80 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (7.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
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 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (7.80 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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。