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Purity: ≥98%
TG003 (TG-003; TG 003) is a novel, potent, selective and ATP-competitive Cdc2-like kinase (Clk) inhibitor with potential anticancer activity. Its IC50 values for Clk1/2/4 inhibition are 20 nM, 200 nM, and 15 nM, respectively. TG003 exhibits no inhibitory action towards PKC, SRPK1, SRPK2, or Clk3.
| Targets |
CLK1 (IC50 = 20 nM); CLK2 (IC50 = 200 nM); CLK4 (IC50 = 15 nM)
TG003 targets the CDC2-like kinase (Clk) family, including Clk1 (IC50=20 nM), Clk2 (IC50=40 nM), Clk3 (IC50=34 nM), Clk4 (IC50=18 nM); it also inhibits dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A, IC50=15 nM) [1] TG003 specifically inhibits Clk1/2/3/4 and regulates pre-mRNA alternative splicing by modulating the activity of these kinases [2] TG003 also targets casein kinase 1 (CK1) isoforms, including CK1δ (IC50=58 nM) and CK1ε (IC50=65 nM) [3] |
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| ln Vitro |
TG003, exhibits the strongest effect on Clk1/Sty and Clk4 (IC50, 15–20 nM) and lesser on Clk2 (200 nM). TG003 suppresses Clk-mediated phosphorylation to prevent SF2/ASF-dependent splicing of β-globin pre-mRNA in vitro. In mammalian cells, it inhibits the phosphorylation of serine/arginine-rich proteins, the dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing[1]. Through modification of TP53 intron 9 alternative splicing, the small drug TG003 increases the endogenous expression of the protein isoforms p53β and p53β[2].
TG003 significantly inhibits the kinase activity of Clk1/2/3/4 in HeLa cells at a concentration of 1 μM, leading to dephosphorylation of SR proteins (splicing factors), thereby altering the alternative splicing patterns of multiple genes, including changes in the ratio of splicing isoforms of CD44 and MAPT genes [1] After treating HCT116 cells with TG003 (1 μM, 48 hours), the protein expression levels of p53β and p53γ (alternative splicing products of the TP53 gene) are significantly upregulated (2.8-fold and 3.2-fold of the control group, respectively); it also enhances cell sensitivity to DNA-damaging agents, with the apoptosis rate increased by 50% compared with the control group [2] In primary mouse dorsal root ganglion (DRG) cells, TG003 inhibits the activity of CK1δ/ε at a concentration of 0.5 μM, reducing the expression level of phosphorylated ERK1/2 in pain-related signaling pathways (decreased by 40%); it has no significant effect on the survival rate of DRG cells (survival rate ≥90% when concentration ≤2 μM) [3] |
| ln Vivo |
TG003 (10 μM) corrects the embryonic defects caused by overactive Clk activity in Xenopus.
TG003 administered intraperitoneally at a dose of 10 mg/kg once daily for 3 consecutive days significantly alleviates behavioral hypersensitivity in mice with complete Freund's adjuvant (CFA)-induced inflammatory pain: 24 hours after administration, the mechanical paw withdrawal threshold of mice increases from 3.2 g to 6.8 g, and the thermal tail flick latency prolongs from 2.1 seconds to 4.3 seconds [3] TG003 administered intraperitoneally at a dose of 15 mg/kg once daily for 5 consecutive days shows an analgesic effect lasting up to 48 hours after the last administration in mice with CFA-induced chronic inflammatory pain, without obvious motor dysfunction [3] |
| Enzyme Assay |
In a reaction mixture containing 200 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 8 mM dithiothreitol, 4 mM EGTA, 1–20 μM ATP, 1 μCi of [γ– 32 P]ATP, 1 μg of synthetic peptide of SF2/ASF RS domain, and 0.1–1 μg of purified kinases in a final volume of 40 μL are used to measure the kinase activity of Clks and SRPKs. Regardless of the concentration of the inhibitor, the final concentration of Me2SO is adjusted to 1%. Half of the reaction mixture is spotted on P81 phosphocellulose membrane after it is incubated for 10 minutes at 30 °C for mammalian recombinant proteins and 25 °C for Xenopus recombinant proteins. The incubation time and substrate and kinase concentrations are among the parameters of the kinase assay that are optimized to preserve linearity. For at least fifteen minutes, the membrane is cleaned with either a 5% phosphoric acid solution or a 5% trichloroacetic solution. A liquid scintillation counter is utilized to quantify the radioactivity [1].
Recombinant Clk1/2/3/4 and DYRK1A kinases were prepared. Different concentrations of TG003 were mixed with kinase solution, ATP substrate, and specific peptide substrate, and incubated at 37°C for 45 minutes; the phosphorylation level of the substrate was determined by radioactive phosphorylation assay to calculate the IC50 value of each kinase [1] Recombinant CK1δ/ε kinases were pre-incubated with gradient concentrations of TG003 for 10 minutes, then ATP and fluorescently labeled substrate peptides were added to initiate the reaction. After incubating at 30°C for 30 minutes, the fluorescence signal intensity was detected by fluorescence resonance energy transfer (FRET) to calculate the kinase activity inhibition rate and IC50 value [3] |
| Cell Assay |
Plate 2x10 5 HeLa or 1.5x10 5 COS-7 cells resuspended in 2 mL of medium on 6-well plates; in some wells, add 2 μL of 10 mM TG003 dissolved in Me2SO (final concentration at 10 mM) or 2 μL of Me2SO. Trypsinized cells are counted every 24 hours for three days, after which the density is determined. After fixing the cells in 1 mL of ice-cold 70% ethanol, they are next cleaned with PBS, incubated for 20 minutes at 37 °C in 1 mL of PBS containing 50 μg/mL propidium iodide and 1 μg/mL DNase-free RNase A, and then subjected to cell cycle analysis[1].
HeLa cells were seeded in 6-well plates (2×10⁵ cells/well) and cultured for 24 hours, then gradient concentrations of TG003 (0.1-5 μM) were added and cultured for another 24 hours; total cellular RNA was extracted, and the splicing products of CD44 and MAPT genes were amplified by reverse transcription-polymerase chain reaction (RT-PCR), and the ratio of splicing isoforms was analyzed by agarose gel electrophoresis [1] HCT116 cells were seeded and cultured for 24 hours, then treated with 1 μM TG003 for 48 hours; cells were collected to extract total protein, which was subjected to SDS-PAGE electrophoresis and membrane transfer, then incubated with antibodies against p53β, p53γ and internal reference protein, and the protein expression level was detected by chemiluminescence; Annexin V-FITC/PI staining was used to detect the cell apoptosis rate by flow cytometry [2] Primary DRG cells were isolated from the spinal cord of neonatal mice, seeded in culture dishes coated with matrix, and cultured for 48 hours before being treated with 0.1-2 μM TG003 for 24 hours; total cellular protein was extracted, and the expression levels of phosphorylated ERK1/2 and total ERK1/2 were detected by Western blot; the cell survival rate was detected by CCK-8 method [3] |
| Animal Protocol |
Intrathecal injection of either TG003 (1-100 pM) or IC261 (0.1-1 nM) dose-dependently decreases mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA.
Xenopus C57BL/6 mice (8-10 weeks old) were subcutaneously injected with complete Freund's adjuvant (CFA) into the right hind paw to establish an inflammatory pain model. Drug administration started 24 hours after modeling; TG003 was dissolved in 5% DMSO + 10% Cremophor EL + 85% normal saline, and administered intraperitoneally at a dose of 10 mg/kg once daily for 3 consecutive days; the mechanical paw withdrawal threshold (von Frey filament method) and thermal tail flick latency (hot plate method) of mice were detected before administration, 24 hours and 48 hours after administration [3] A high-dose group was also set up, where CFA-modeled mice were given TG003 15 mg/kg intraperitoneally once daily for 5 consecutive days; behavioral indicators were detected 24 hours and 48 hours after the last administration, and mice were sacrificed at the end of the experiment to detect CK1δ/ε activity in DRG tissues [3] |
| Toxicity/Toxicokinetics |
In in vitro cell experiments, at a concentration ≤2 μM, TG003 showed no significant cytotoxicity to HeLa, HCT116, and primary DRG cells, with cell viability ≥85% [1][2][3]. When TG003 was administered intraperitoneally at a dose of 15 mg/kg (for 5 consecutive days), no significant toxic reactions, such as significant weight loss, anorexia, or motor dysfunction, were observed in mice with inflammatory pain [3].
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| References |
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| Additional Infomation |
TG003 is the first highly selective Clk family inhibitor developed. It regulates the alternative splicing of precursor mRNA by inhibiting Clk-mediated phosphorylation of splicing factors, providing a tool drug for studying splicing-related diseases [1]. TG003 can regulate the expression ratio of p53β and p53γ by regulating the alternative splicing of the TP53 gene, thereby modulating the way cells respond to DNA damage and providing a potential target for tumor treatment [2]. TG003 blocks the transmission of pain signaling pathways by inhibiting CK1δ/ε kinase activity, showing significant analgesic effects in inflammatory pain mouse models, providing a new candidate drug direction for the treatment of chronic pain [3].
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| Molecular Formula |
C13H15NO2S
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| Molecular Weight |
249.33
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| Exact Mass |
249.082
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| Elemental Analysis |
C, 62.62; H, 6.06; N, 5.62; O, 12.83; S, 12.86
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| CAS # |
719277-26-6
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| Related CAS # |
300801-52-9;719277-26-6
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| PubChem CID |
1893668
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| Appearance |
white solid powder
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| LogP |
2.381
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
17
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| Complexity |
329
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(/C=C1SC2=CC=C(OC)C=C2N\1CC)=O
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| InChi Key |
BGVLELSCIHASRV-QPEQYQDCSA-N
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| InChi Code |
InChI=1S/C13H15NO2S/c1-4-14-11-8-10(16-3)5-6-12(11)17-13(14)7-9(2)15/h5-8H,4H2,1-3H3/b13-7-
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| Chemical Name |
(1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (8.34 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (8.34 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (8.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.0107 mL | 20.0537 mL | 40.1075 mL | |
| 5 mM | 0.8021 mL | 4.0107 mL | 8.0215 mL | |
| 10 mM | 0.4011 mL | 2.0054 mL | 4.0107 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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