CK2/casein kinase 2

IQA [[5-oxo-5,6-dihydro-indolo(1,2-a)quinazolin-7-yl]acetic acid] is a novel ATP/GTP site-directed inhibitor of CK2 ('casein kinase 2'), a pleiotropic and constitutively active protein kinase whose activity is abnormally high in transformed cells. The K (i) value of IQA (0.17 microM) is lower than those of other CK2 inhibitors reported so far. Tested at 10 microM concentration in the presence of 100 microM ATP, IQA almost suppresses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of 44 protein kinases and on phosphoinositide 3-kinase. In comparison, other CK2 inhibitors, notably apigenin and quercetin, are more promiscuous. The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner. IQA has been co-crystallized with maize CK2alpha, which is >70% identical with its human homologue, and the structure of the complex has been determined at 1.68 A (1 A=0.1 nm) resolution. The inhibitor lies in the same plane occupied by the purine moiety of ATP with its more hydrophobic side facing the hinge region. Major contributions to the interaction are provided by hydrophobic forces and non-polar interactions involving the aromatic portion of the inhibitor and the hydrophobic residues surrounding the ATP-binding pocket, with special reference to the side chains of V53 (Val53), I66, M163 and I174. Consequently, mutants of human CK2alpha in which either V66 (the homologue of maize CK2alpha I66) or I174 is replaced by alanine are considerably less sensitive to IQA inhibition when compared with wild-type. These results provide new tools for deciphering the enigmatic role of CK2 in living cells and may pave the way for the development of drugs depending on CK2 activity [1].

The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner[2].

Source and purification of protein kinases[2]
Native CK1 and CK2 were purified from rat liver. G-CK (Golgi CK), purified from lactating mammary gland of rats, was a gift from Dr A. M. Brunati (University of Padova, Padova, Italy). Protein tyrosine kinases Lyn, c-Fgr and Syk (also termed TPK-IIB) were purified from rat spleen. Human recombinant α and β subunits of CK2 were expressed in Escherichia coli and the holoenzyme was reconstituted and purified as described previously. The CK2α mutant V66A was obtained by the method described in using the oligonucleotide 5 -AAAAAGTTGCTGTTAAAAT-3 . The S51G and I174A mutants were obtained using the QuikChange sitedirected mutagenesis kit. Each mutant was generated using two synthetic oligonucleotide primers, each complementary to opposite strands of human α cDNA inserted in pT7-7 vector, namely 5 -GGCCGAGGTAAATACGGTGAAGTATTTGAAGCC-3 and 5 -GCACAGAAAGCTACGACTAGCAGACTGGGGTTTGGC-3 for S51G and I174A respectively. Mutants were confirmed by sequencing and used to transform competent E. coli BL21(DE3) cells. Expression and purification of α V66A, α S51G and α I174A were performed as described. Catalytic α subunits were dialysed against 25 mM Tris/HCl (pH 7.5) containing 50% (v/v) glycerol and were stored at − 20 ◦C. Saccharomyces cerevisiae piD261 was kindly provided by Dr S. Facchin.

---

Phosphorylation experiments[2]
All protein kinase activities were linear with respect to time and enzyme concentration in each incubation. Phosphorylation conditions and evaluation of the phosphate incorporated were as described in [20] for CK2, CK1, G-CK, piD261 and the tyrosine kinases Lyn, c-Fgr and Syk. All other specificity assays were performed using an automated multichannel pipette system at room temperature (25 ◦C) in a total assay volume of 25 µl. In detail, to plates containing 0.5 µl of compounds, DMSO controls (without inhibitors) or acid blanks, 15 µl of an enzyme mixture containing the enzyme and the peptide/protein substrate in buffer was added. Compounds were preincubated in the presence of the enzyme and the peptide/protein substrate for 5 min before initiation of the reaction by adding 10 µl of ATP (final concentration of 100 µM for all enzymes). Assays were performed for 40 min at room temperature before termination by the addition of 5 µl of orthophosphoric acid. After mixing, 10 µl of the reaction mixture was spotted on to P30 filtermats, which were then washed in phosphoric acid. The filters were subjected to washes of 1 × 3 min, followed by 3 × 8 min.

The human leukaemia Jurkat T-cell line was maintained in RPMI 1640, supplemented with 10% (v/v) foetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin, in an atmosphere containing 5% CO2. Before treatment, cells were washed, resuspended at a density of approx. 106 cells/ml in a medium containing 1% foetal calf serum, and then incubated at 37 ◦C in the presence of the compound at the indicated concentration for different periods of time, as described in the Figure legends. Control cells were treated with equal amounts of the solvent. At the end of the incubations, cells were centrifuged, washed and lysed by the addition of ice-cold hypo-osmotic buffer consisting of 10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, protease inhibitor cocktail , 10 mM NaF and 1 µM okadaic acid. Cytosol and the nuclear extract were prepared as described. Immunoprecipitation of HS1 (haematopoietic cell-specific protein 1) and Western-blot analysis were performed for the cytosolic fraction using an anti-HS1 total protein antiserum as described. The assay of CK2 activity in lysates of cells pretreated with increasing concentrations of IQA was performed as described previously[2].

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (Ki = 0.4 microM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (Ki = 0.040 microM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (Ki = 0.22 microM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (Ki = 0.17 microM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs[1].

[1]. Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA). Biochem J. 2003 Sep 15; 374(Pt 3): 639–646.
[2]. Development and exploitation of CK2 inhibitors. Mol Cell Biochem . 2005 Jun;274(1-2):69-76. doi: 10.1007/s11010-005-3079-z
[3]. Liu J, et al. Cascade Reaction of Morita-Baylis-Hillman Acetates with 1,1-Enediamines or Heterocyclic Ketene Aminals: Synthesis of Highly Functionalized 2-Aminopyrroles. J Org Chem. 2019 Feb 15;84(4):1797-1807.

C17H12N2O3

292.29

292.085

C, 69.86; H, 4.14; N, 9.58; O, 16.42

391670-48-7

447682

Solid powder

1.46

2.973

2

3

2

22

481

0

C1=CC=C2C(=C1)C(=C3N2C4=CC=CC=C4C(=O)N3)CC(=O)O

INSBKYCYLCEBOD-UHFFFAOYSA-N

InChI=1S/C17H12N2O3/c20-15(21)9-12-10-5-1-3-7-13(10)19-14-8-4-2-6-11(14)17(22)18-16(12)19/h1-8H,9H2,(H,18,22)(H,20,21)

2-(5-oxo-5,6-dihydroindolo[1,2-a]quinazolin-7-yl)acetic acid

CGP 029482; CGP029482; CGP-029482

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)

Typically soluble in DMSO (e.g. > 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)