Quercetagetin inhibits PIM2, PKA, and RSK2 at IC50s of 3.45, 21.2, and 2.82 μM, respectively [2]. Quercetagetin (0.1, 1, 10, and 100 μM, 72 hours) inhibited the proliferation of RWPE2 prostate cancer cells with an average ED50 of 3.8 μM [2].

Quercetagetin dramatically reduces the growth of UVB-induced skin cancer. When applied topically to mouse skin, Quercetagetin at concentrations of 4 or 20 nmol decreased tumor incidence by 32.0% and 46.7%, respectively [3].

Recombinant pim Kinases and Kinase Assays[1]
We prepared recombinant PIM1 and PIM2 as glutathione S-transferase (GST) fusions in Escherichia coli, as described. For the inhibitor screening assays, a solid-phase kinase assay was developed based on our demonstration that PIM1 is a potent kinase for phosphorylating BAD on Ser112. Ninety-six-well flat-bottomed plates were coated overnight at 4°C with recombinant GST-BAD [1 μg/well in HEPES buffer: 136 mmol/L NaCl, 2.6 mmol/L KCl, and 20 mmol/L HEPES (pH 7.5)]. The plates were then blocked for 1 h at room temperature with 10 μg/mL bovine serum albumin in HEPES buffer. The blocking solution was then removed and 5 μL of each inhibitor, dissolved in 50% DMSO, were added to each well. Then, 100 μL of kinase buffer [20 mmol/L MOPS (pH 7.0), 12.5 mmol/L MgCl2, 1 mmol/L MnCl2, 1 mmol/L EGTA, 150 mmol/L NaCl, 10 μmol/L ATP, 1 mmol/L DTT, and 5 mmol/L β-glycerophosphate] containing 25 ng recombinant GST-PIM1 kinase were added to each well. The final concentration of each inhibitor was ∼10 μmol/L. The plate was placed on a gel slab dryer prewarmed to 30°C, and the kinase reaction was allowed to proceed. The reaction was stopped after 60 min by removal of the reaction buffer, followed by the addition of 100 μL of HEPES buffer containing 20 mmol/L EDTA to each well. Phosphorylated GST-BAD was detected by an ELISA reaction, using as first antibody a monoclonal anti–phospho-BAD(S112) antibody, a secondary goat anti-mouse IgG-peroxidase conjugated antibody, and Turbo-TMB peroxidase substrate. The level of phosphorylated GST-BAD present was proportional to the absorbance at 450 nm.[1]
For quantitative and kinetic studies of inhibitors against various BAD(S112) kinases, a solution phase assay was used. A biotinylated peptide based on the PIM1 phosphorylation site of human BAD was synthesized (GGAGAVEIRSRHSSYPAGTE) and used as the assay substrate. Recombinant GST-PIM1 (25 ng/reaction) was preincubated with various concentrations of inhibitors in the previous kinase buffer (final volume 100 μL). The reaction proceeded by addition of substrate peptide, followed by incubation for 5 min in a 30°C water bath. The reaction was terminated by transferring the mixture to a streptavidin-coated 96-well plate containing 100 μL/well of 40 mmol/L EDTA. The biotinylated peptide substrate was allowed to bind to the plate at room temperature for 10 min. The level of phosphorylation was then determined by ELISA as described above. Curve fitting and enzyme analyses were done using GraphPad Prism version 4.00 for Windows. For the additional BAD(S112) kinases [PIM2, RSK2 (ribosomal S6 kinase 2), and PKA (cyclic AMP–dependent protein kinase)], reaction components were as described above. As with the PIM1 assays, an ATP concentration of 10 μmol/L was used. Furthermore, with each kinase, linear reaction velocities for the duration of the reaction were confirmed (data not shown).[1]
To further assess the specificity of quercetagetin as a PIM1 inhibitor, its activity against a panel of serine-threonine kinases was also studied through a commercial kinase inhibitor profiling service (KinaseProfiler; Upstate Biotechnology, Charlottesville, VA). All KinaseProfiler assays were conducted using 10 μmol/L ATP concentrations.[1]

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Small-Molecule Library Screening[1]
We obtained a library of 1,200 compounds that had structural affinity to known kinase inhibitors. The entire library was screened once with our solid-phase ELISA kinase assay, with each compound at ∼10 μmol/L concentration. Positive hits were rescreened at the same concentration. Compounds that had reproducible activity at 10 μmol/L were then screened at a range of concentrations from 0.001 to 300 μmol/L.

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Measurement of PIM1 Kinase Activity in Cells[1]
RWPE2 cell pools, stably infected with empty retrovirus or pim-1–encoding retrovirus, were seeded in six-well plates at 5 × 105 cells per well. After 18 h, the normal supplemented keratinocyte medium was removed and replaced with supplement-free keratinocyte medium. Cells were then incubated for an additional 20 h. Quercetagetin, or an equivalent volume of DMSO, was added to the cells 3 h before the end of the starvation period. At the conclusion of the starvation period, the cells were washed twice with PBS and subsequently lysed in a denaturing buffer with protease, phosphatase inhibitors.

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Cloning, Expression, Purification, and Crystallization of PIM1[1]
The production, purification, and characterization of recombinant 6His-tagged PIM1 proteins for crystallography have been described previously. To obtain cocrystals of complexes of the protein with ligands, the protein solution was initially mixed with the compound (dissolved in DMSO) at a final compound concentration of 1 mmol/L and then set up for crystallization. The protein was crystallized by a sitting-drop, vapor-diffusion experiment in which equal volumes of protein (10–15 mg/mL concentration) and reservoir solution [0.4–0.9 mol/L sodium acetate, 0.1 mol/L imidazole (pH 6.5)] were mixed and allowed to equilibrate against the reservoir at 4°C. The crystals routinely grew to a size of 200 × 200 × 800 μm in ∼2 to 3 days.

Cell Viability Assay[2]
Cell Types: RWPE2 prostate cancer cells
Tested Concentrations: 0.1, 1, 10, and 100 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Inhibited growth of RWPE2 prostate cancer cells with average ED50 is 3.8 μM.

Animal/Disease Models: SKH-1 hairless mice model[3]
Doses: 4 or 20 nmol
Route of Administration: Topical application; 28 weeks
Experimental Results: Inhibited UVB-induced skin tumorigenesis in SKH-1 hairless mice models. Delayed the development of tumors and decreased tumor volumes in an SKH-1 hairless mice model.

[1]. Yang X, et al. Isolation and identification of an antioxidant flavonoid compound from citrus-processing by-product. J Sci Food Agric. 2011 Aug 15;91(10):1925-7.
[2]. Holder S, et al. Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase. Mol Cancer Ther. 2007 Jan;6(1):163-72.
[3]. Baek S, et al. Structural and functional analysis of the natural JNK1 inhibitor quercetagetin. J Mol Biol. 2013 Jan 23;425(2):411-23.

Quercetagetin is a hexahydroxyflavone that is flavone substituted by hydroxy groups at positions 3, 5, 6, 7, 3' and 4' respectively. It has a role as an antioxidant, an antiviral agent and a plant metabolite. It is a member of flavonols and a hexahydroxyflavone. It is functionally related to a quercetin.
Quercetagetin is a natural product found in Citrus reticulata, Cupressus sempervirens, and other organisms with data available.

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The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3,3',4',5,6,7-hydroxyflavone) was identified as a moderately potent, ATP-competitive inhibitor (IC(50), 0.34 micromol/L). Resolution of the crystal structure of PIM1 in complex with quercetagetin or two other flavonoids revealed a spectrum of binding poses and hydrogen-bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared with PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED(50), 5.5 micromol/L). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked PIM1 kinase activity. Furthermore, the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.[1]

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c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3',4',5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model. Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.[2]

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Results: The major flavonoid isolated from C. unshiu peel was identified as quercetagetin. The structure of the compound was determined by tandem mass spectrometry and ultraviolet spectroscopy. Its antioxidant activity was assessed by assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical and intracellular reactive oxygen species (ROS) scavenging and DNA damage inhibition. Quercetagetin showed strong DPPH radical-scavenging activity (IC₅₀ 7.89 µmol L⁻¹) but much lower hydroxyl radical-scavenging activity (IC₅₀ 203.82 µmol L⁻¹). Furthermore, it significantly reduced ROS in Vero cells and showed a strong protective effect against hydrogen peroxide-induced DNA damage.[3]
Conclusion: The results of this study suggest that quercetagetin could be used in the functional food, cosmetic and pharmaceutical industries.

C₁₅H₁₀O₈

318.24

318.038

90-18-6

5281680

Typically exists as light yellow to yellow solids at room temperature

1.912 g/cm3

732.4ºC at 760 mmHg

>300ºC

280.3ºC

1.863

1.69

151.59

O1C(=C(C(C2C(=C(C(=C([H])C1=2)O[H])O[H])O[H])=O)O[H])C1C([H])=C([H])C(=C(C=1[H])O[H])O[H]

ZVOLCUVKHLEPEV-UHFFFAOYSA-N

InChI=1S/C15H10O8/c16-6-2-1-5(3-7(6)17)15-14(22)13(21)10-9(23-15)4-8(18)11(19)12(10)20/h1-4,16-20,22H

2-(3,4-dihydroxyphenyl)-3,5,6,7-tetrahydroxychromen-4-one

6-Hydroxyquercetin; 6-Hydroxyquercetin; Quercetagenin; 3,3',4',5,6,7-Hexahydroxyflavone; UNII-SV68G507VO; 3,5,6,7,3',4'-Hexahydroxyflavone; 2-(3,4-dihydroxyphenyl)-3,5,6,7-tetrahydroxychromen-4-one;

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 : ~125 mg/mL (~392.79 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.54 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 20.8 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: ≥ 2.08 mg/mL (6.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)