SERCA (sarco/endoplasmic reticulum Ca2+-ATPase)

Treatment with CDN1163 (10 μM; 24 hours; rat cardiomyocytes) decreases nuclear NFATc and resistin expression produced by high hyperglycemia and, in a time-dependent way, enhances AMPKα phosphorylation [2].
CDN1163 markedly abolished glucose-stimulated NFATc nuclear translocation. Likewise, in the presence of CDN1163 high glucose-induced resistin and nuclear NFATc expression were significantly reduced while the phosphorylation of AMPKα is increased in a time-dependent manner, indicating that CDN1163-mediated activation of Serca2a function affects resistin and NFATc expression patterns in a similar manner as Serca2a gene transfer. Interestingly, CDN1163 also reduced NFATc-mediated resistin promoter luciferase activity[2].
CDN1163 Increases SERCA2 Ca2+-ATPase Activity, Decreases ER Stress-induced Cell Death in Vitro.[1]

Male ob/ob and lean ob/+ mice were given 50 mg/kg of CDN1163 intraperitoneally (i.p.) for five days. This treatment improved hepatic Ca2+ transport activity, decreased endoplasmic reticulum (ER) stress-induced cell death in vitro, and increased SERCA2 Ca2+-ATPase activity. In ob/ob mice, CDN1163 lowers blood glucose levels, enhances metabolic parameters and gluconeogenesis gene expression, reverses hepatic steatosis, prevents ER stress and ER stress-induced apoptosis, and boosts mitochondrial efficiency [1].

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Ob/ob mice were treated with CDN1163 for 4 weeks as indicated in the methods and the cardiac levels of NFATc and resistin were then analyzed by immunoblotting. Indeed, hearts from CDN1163-treated mice showed significant decrease in resistin (Fig. 8F,G) and nuclear NFATc (Fig. 8F,H) protein expression compared with vehicle-treated mice. Consistent with the decline in resistin expression, CDN1163 treatment increased AMPKα activity/phosphorylation in ob/ob mice hearts compared to vehicle-treated (Fig. 8F,I). Altogether, these results demonstrate that CDN1163 is able to regulate resistin expression in vitro and in vivo, validating the pharmacological activation of Serca2a as a treatment for hyper-resistinemic conditions.[2]

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.[3]

Western Blot analysis [2]
Cell Types: rat cardiomyocytes (H9c2)
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: High glucose-induced resistin and nuclear NFATc expression were Dramatically diminished. Phosphorylation of AMPKα increases in a time-dependent manner.

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ER Stress Cell Viability Assay[1]
Cells were grown in 96-well plates (n = 6) and exposed to CDN1163 (10 μm) or vehicle (DMSO) for 2 h followed by the addition of 200 μm H2O2 compared with untreated controls. H2O2 activates the unfolded protein response and is an inducer of ER stress-promoted apoptosis. After 16 h of incubation, cell viability was measured using CellTiter-Glo® Luminescent Cell Viability Assay[1].

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Rat cardiac myocyte cells (H9c2) were grown in DMEM supplemented with 10% FBS and 1X cocktail of pen/strep antibiotics. 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-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)/BAPTA-AM (2 μM), or Ca2+ (4 mM) in low glucose (5 mM) or high glucose (25 mM) to mimic diabetic condition for the indicated times (20 mM mannitol is added to verify glucose induced osmotic effects). Cells were lysed and harvested for real-time-PCR and western analysis.[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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.80 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 (7.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (7.80 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.)