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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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CP-91149 is a selective glycogen phosphorylase (GP) inhibitor with IC50 of 0.13 μM. |
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Targets
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GP |
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IC50 |
0.13 μM [1] |
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In Vitro
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CP-91149 displays 200-fold higher inhibitory activity against human liver glycogen phosphorylase a (HLGPa) than caffeine (IC50 = 26 μM). CP-91149 (10-100 μM) inhibits glucagon-stimulated glycogenolysis in isolated rat hepatocytes in a dose-dependent manner, and in primary human hepatocytes with IC50 of ~2.1 μM. [1] CP-91149 also potently inhibits the activities of human muscle phosphorylase a and b with IC50 of 0.2 μM and ~0.3 μM, respectively. CP-91149 treatment at 2.5 μM induces inactivation of phosphorylase and sequential activation of glycogen synthase in hepatocytes, and increases glycogen synthesis by 7-fold at 5 mM glucose and by 2-fold at 20 mM glucose. CP-91149 can partially counteract the effects of phosphorylase overexpression. [2] CP-91149 also potently inhibits brain GP with IC50 of 0.5 μM in A549 cells. CP-91149 treatment at 10-30 μM causes significant glycogen accumulation in A549 and HSF55 cells. CP-91149 treatment increases G1-phase cells with a significant reduction of the S-phase population in HSF55 cells, correlated with increased expression of p21 and p27. [3] CP-91149 also promotes the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. [4] |
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In Vivo
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Oral administration of CP-91149 to diabetic ob/ob mice at 25-50 mg/kg causes rapid (3 hours) glucose lowering by 100-120 mg/dl without producing hypoglycemia, resulting from inhibition of glycogenolysis in vivo. CP-91149 treatment does not lower glucose levels in normoglycemic, nondiabetic mice. [1] In the non-fasted Goto-Kakizaki (GK) rats, administration of CP-91149 in combination with CS-917 suppresses hepatic glycogen reduction by CS-917 and decreases plasma glucose more than single administration of CS-917. [5] |
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Clinical Trials
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Features
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Combination Therapy
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Description
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HLGPa inhibition by CP-91149 is synergistic with caffeine and theophylline, as caffeine or theophylline increases the potency (IC50) of CP-91149 from 1 μM to 0.14 μM. [1] |
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Protocol
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Kinase Assay
[1]
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| Phosphorylase enzyme assay |
Human liver glycogen phosphorylase a (HLGPa, 85 ng) activity is measured in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate at 22°C in 100 μL of buffer containing 50 mM Hepes (pH 7.2), 100 mM KCl, 2.5 mM EGTA, 2.5 mM MgCl2, 0.5 mM glucose-1-phosphate, and 1 mg/mL glycogen. Phosphate is measured at 620 nm, 20 minutes after the addition of 150 μL of 1 M HCl containing 10 mg/mL ammonium molybdate and 0.38 mg/mL malachite green. Increasing concentrations of CP-91149 are added to the assay in 5 μL of 14% DMSO. |
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Cell Assay
[3]
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| Cell Lines |
HSF55 and T98G |
| Concentrations |
Dissolved in DMSO, final concentrations ~50 μM |
| Incubation Time |
72 hours |
| Methods |
Cells are exposed to various concentrations of CP-91149 for 72hours. Viability is determined with manual cell counts following staining with trypan blue exclusion assay. Cells are fixed with 70% ethanol. DNA is stained with propidium iodide and the intensity of fluorescence is measured using a Becton-Dickinson flow cytometer at 488nm for excitation and at 650nm for emission. The cell cycle profile is analyzed using Modifit’s Sync Wizard. |
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Animal Study
[1]
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| Animal Models |
Obese, diabetic male C57BL/6J-Lep(ob/ob) mice and their lean, nondiabetic C57BL/6J-?/+ littermates |
| Formulation |
Formulated in vehicle consisting of either 0.25% (wt/vol) methyl cellulose in water or 0.1% Pluronic P105 Block Copolymer Surfactant in 0.1% saline |
| Doses |
~50 mg/kg |
| Administration |
Orally |
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References |
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[1] Martin WH, et al. Proc Natl Acad Sci U S A, 1998, 95(4), 1776-1781.
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[2] Aiston S, et al. J Biol Chem, 2001, 276(26), 23858-23866.
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[3] Schnier JB, et al. Biochem Biophys Res Commun, 2003, 309(1), 126-134.
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[4] Lerín C, et al. Biochem J, 2004, 378(Pt 3), 1073-1077.
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[5] Yoshida T, et al. J Pharmacol Sci, 2011, 115(3), 329-335.
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