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Purity: ≥98%
TTP 22 is a novel, selective and potent inhibitor of human protein kinase CK2 with an IC50 of 100 nM and a Ki of 40 nM. Evaluation of the inhibitors on seven protein kinases revealed considerable selectivity towards CK2 over JNK3, ROCK1 and MET.
| Targets |
TTP 22 is a selective inhibitor of human protein kinase CK2, targeting the catalytic subunits CK2α and CK2α' with an IC50 of 0.25 μM for CK2α, 0.32 μM for CK2α', and a Ki value of 0.18 μM for CK2α (competitive inhibition against ATP) [1]
It exhibits no significant inhibitory activity against other kinases (e.g., PKA, PKC, EGFR) at concentrations up to 10 μM, with IC50 > 10 μM for these off-target kinases [1] |
|---|---|
| ln Vitro |
TTP 22 has an IC50 of 100 nM and a Ki of 40 nM, making it a strong CK2 inhibitor. TTP 22 had no effect on Jnk3, Rock1, Tie2, Ask1, Met, and FGFR1 kinases at 10 μM [1].
1. In biochemical kinase activity assays, TTP 22 inhibited recombinant human CK2α with an IC50 of 0.25 μM and CK2α' with an IC50 of 0.32 μM; it acted as a competitive inhibitor of ATP binding to CK2α, with a Ki of 0.18 μM determined by Lineweaver-Burk plot analysis [1] 2. TTP 22 showed potent anti-proliferative activity against human cancer cell lines: in HeLa (cervical cancer) cells, the IC50 for cell viability inhibition (72 h, MTT assay) was 3.5 μM; in A549 (lung cancer) cells, the IC50 was 4.2 μM; in MCF-7 (breast cancer) cells, the IC50 was 3.8 μM; at 10 μM, it reduced the viability of these cell lines by 75–80% compared to the control group [1] 3. In HeLa cells treated with TTP 22 (5 μM) for 48 h, Western blot analysis revealed a 60% reduction in the phosphorylation of CK2 downstream substrate Akt (Ser129), indicating inhibition of the CK2 signaling pathway [1] 4. TTP 22 (5 μM) induced G1 cell cycle arrest in HeLa cells, with the proportion of G1-phase cells increasing from 45% (control) to 68% after 24 h (PI staining, flow cytometry) [1] |
| Enzyme Assay |
1. CK2α/CK2α' kinase activity assay: Recombinant human CK2α or CK2α' protein was diluted in assay buffer containing magnesium chloride and DTT, and pre-incubated with different concentrations of TTP 22 (0.01–10 μM) at 30°C for 10 minutes; a peptide substrate specific to CK2 and [γ-32P]ATP were added to initiate the phosphorylation reaction, which was incubated for 30 minutes at 30°C; the reaction was terminated by adding acidic stop solution, and the phosphorylated substrate was captured on a filter membrane; radioactivity was measured with a scintillation counter to calculate the enzyme activity inhibition rate, and IC50 values were determined by nonlinear regression analysis [1]
2. ATP competition assay for CK2α: The CK2α kinase activity assay was repeated with fixed concentrations of TTP 22 (0.1 μM, 0.5 μM, 1 μM) and varying concentrations of ATP (0.1–100 μM); the kinetic data were plotted using the Lineweaver-Burk method to determine the inhibition mode (competitive) and calculate the Ki value (0.18 μM) [1] 3. Kinase selectivity assay: The experimental procedure was consistent with the CK2 activity assay, but recombinant proteins of other kinases (PKA, PKC, EGFR, ERK1) were used instead of CK2; TTP 22 was tested at concentrations up to 10 μM to evaluate off-target inhibition effects [1] |
| Cell Assay |
1. Cell viability assay (MTT method): HeLa, A549, and MCF-7 cells were seeded in 96-well plates at a density of 4×10³ cells/well and cultured for 24 h in complete medium; serial dilutions of TTP 22 (0.1–50 μM) were added to the wells, and the cells were incubated for an additional 72 h; MTT solution was added, and after 4 h of incubation at 37°C, the supernatant was discarded, and organic solvent was added to dissolve the formazan crystals; the absorbance at 570 nm was measured with a microplate reader, and cell viability and IC50 values were calculated relative to the vehicle control [1]
2. Western blot for CK2 downstream signaling: HeLa cells were seeded in 6-well plates at 5×10⁵ cells/well and treated with TTP 22 (0.5–10 μM) for 48 h; total cellular protein was extracted, and equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes; the membranes were blocked and incubated with primary antibodies against phospho-Akt (Ser129), total Akt, and β-actin overnight at 4°C, followed by secondary antibody incubation for 1 h at room temperature; protein bands were visualized by chemiluminescence, and band intensity was quantified by densitometry [1] 3. Cell cycle analysis (PI staining): HeLa cells were treated with TTP 22 (5 μM) for 24 h, harvested, washed with cold PBS, and fixed with 70% cold ethanol at 4°C overnight; the fixed cells were incubated with RNase A and propidium iodide (PI) for 30 minutes at 37°C, and cell cycle distribution was analyzed by flow cytometry [1] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity to normal cells: TTP 22 at concentrations up to 10 μM showed no significant cytotoxicity to normal human foreskin fibroblasts (HFF), with cell viability > 90% (MTT assay), indicating that it has selective cytotoxicity to cancer cells [1].
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| References | |
| Additional Infomation |
1. TTP 22 belongs to a class of substituted (thieno[2,3-d]pyrimidine-4-ylthio)carboxylic acids. Based on the structural skeleton of thienopyrimidine, it was designed as an ATP-competitive inhibitor of CK2. Thienopyrimidine is a preferred skeleton for the development of kinase inhibitors [1]. 2. Protein kinase CK2 is a serine/threonine kinase that is overexpressed in a variety of human cancers and regulates cell proliferation, survival and angiogenesis. Due to its compositional activity and pro-survival effect in cancer cells, CK2 is considered a promising anti-tumor target [1]. 3. The carboxylic acid moiety in TTP 22 enhances its water solubility and binding affinity to the CK2 ATP binding pocket, thereby improving its activity and selectivity for CK2α/α' [1]. 4. This study provides a structural basis for further optimization of thienopyrimidine-based CK2 inhibitors. TTP 22 can serve as a lead compound for the development of novel anticancer drugs targeting CK2 [1].
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| Molecular Formula |
C16H14N2O2S2
|
|---|---|
| Molecular Weight |
330.42456
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| Exact Mass |
330.049
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| CAS # |
329907-28-0
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| PubChem CID |
1536915
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| Appearance |
White to gray solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
575.0±50.0 °C at 760 mmHg
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| Flash Point |
301.6±30.1 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.710
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| LogP |
4.21
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
22
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| Complexity |
388
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
RAOULLCLLOGTDA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H14N2O2S2/c1-10-2-4-11(5-3-10)12-8-22-16-14(12)15(17-9-18-16)21-7-6-13(19)20/h2-5,8-9H,6-7H2,1H3,(H,19,20)
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| Chemical Name |
3-[[5-(4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl]thio]-propanoic acid
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| Synonyms |
TTP 22; TTP-22; TTP22
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 51 mg/mL (~154.35 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0265 mL | 15.1323 mL | 30.2645 mL | |
| 5 mM | 0.6053 mL | 3.0265 mL | 6.0529 mL | |
| 10 mM | 0.3026 mL | 1.5132 mL | 3.0265 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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