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Purity: ≥98%
CLK-IN-T3 is a novel, potent and highly selective CLK (CDC-like kinase) inhibitor with high specificity to CLK1-3 protein isoforms and with IC50s of 0.67 nM, 15 nM, and 110 nM for CLK1, CLK2, and CLK3 protein kinases, respectively. CLK-IN-T3 has anti-cancer activity.
| Targets |
CLK1 (IC50 = 0.67 nM); CLK2 (IC50 = 15 nM); CLK3 (IC50 = 110 nM); DYRK1A (IC50 = 260 nM); DYRK1B (IC50 = 230 nM)
CDC-like kinase 1 (CLK1, IC50 = 0.67 nM), CDC-like kinase 2 (CLK2, IC50 = 15 nM), CDC-like kinase 3 (CLK3, IC50 = 110 nM) DYRK1A and DYRK1B (inhibition approximately 200–300 times weaker than CLK isoforms)[1] |
|---|---|
| ln Vitro |
CLK-IN-T3 suppresses the activity of DYRK1A (IC50=260 nM) and DYRK1B (IC50=230 nM).[1]
CLK-IN-T3 (0.1-10.0 µM; 24 hours) results in a long-lasting (24 hours) mild cell cycle arrest at the G2/M boundary[1]. CLK-IN-T3 (0.5-1.0 µM; 6 hours) reduces the phosphorylation of SR proteins that are targeted by CLK, while CLK proteins slightly increase[1]. CLK-IN-T3 inhibits CLK1, CLK2, and CLK3 with IC50 values of 0.67 nM, 15 nM, and 110 nM, respectively, in enzymatic assays. In cell-based assays, short-term exposure (6 h) to CLK-IN-T3 does not cause cytotoxicity or apoptosis even at high concentrations. Long-term exposure (>24 h) results in mild cell cycle arrest at the G2/M boundary. CLK-IN-T3 reduces phosphorylation of SRSF1, SRSF4, and SRSF6, which are canonical CLK targets in exon recognition. RNA-Seq analysis shows that CLK-IN-T3 induces dose-dependent alternative splicing events, primarily skipped exons (SE) and retained introns (RI), in HCT116 and 184hTERT cells. CLK-IN-T3 also promotes dose-dependent formation of conjoined gene (CG) transcripts through splicing between adjacent genes on the same strand. siRNA knockdown of CLK2-associated genes (e.g., SRRM1/2, SRSF proteins) increases CG formation, supporting the role of CLK in suppressing CG transcription.[1] |
| Enzyme Assay |
Kinase assays for CLK1-3, DYRK1A, and DYRK1B were performed using a luminescence-based kinase assay system.
The kinase buffer contained HEPES (pH 7.5), magnesium acetate, DTT, BSA, and Tween-20. Reaction mixtures contained specific concentrations of each kinase and a peptide substrate labeled with a fluorescent donor. After incubating the enzyme, peptide, and compound for 10 minutes, ATP was added to initiate the reaction, followed by incubation for 45 minutes. Reactions were terminated with EDTA, and phosphorylated substrates were detected using Europium-labeled antibodies against phosphorylated peptides. Fluorescence signals were measured, and IC50 values were calculated using curve-fitting software.[1] |
| Cell Assay |
Cell Line: HCT-116 cells
Concentration: 0.1, 0.5, 1.0, 5.0, 10.0 µM Incubation Time: 24 hours Result: Resulted in mild cell cycle arrest at the G2/M boundary with long-duration (24 h). HCT116 (colorectal cancer) and 184hTERT (immortalized mammary epithelial) cells were cultured under mycoplasma-free conditions. For splicing analysis, cells were treated with increasing concentrations of CLK-IN-T3 for 6 hours to avoid cytotoxicity or cell cycle effects. Total RNA was extracted and used for strand-specific or non-stranded RNA-Seq library preparation. Libraries were sequenced on an Illumina platform, and reads were aligned to the human reference genome. Alternative splicing events were quantified using MISO and VAST-tools. For validation of conjoined genes, targeted PCR sequencing and PacBio long-read sequencing were performed. For siRNA experiments, HCT116 cells were transfected with siRNAs targeting CLK family members or motif-enriched factors using lipid-based transfection reagents. Knockdown efficiency was assessed by qPCR, and effects on CG formation were evaluated by targeted RNA-Seq of a panel of CG events.[1] |
| ADME/Pharmacokinetics |
CLK-IN-T3 exhibited good cell permeability and stability in the culture medium (Supplementary Table 3). [1]
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| Toxicity/Toxicokinetics |
Short-term (6 hours) exposure to CLK-IN-T3 did not induce cytotoxicity or apoptosis, even at high concentrations. Long-term exposure (>24 hours) caused slight cell cycle arrest at the G2/M phase, but no significant cytotoxicity was observed within the tested concentration range.
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| References | |
| Additional Infomation |
CLK-IN-T3 is a novel, highly potent, and selective small molecule inhibitor of CLK1-3, with a structure drastically different from previous inhibitors such as KH-CB19. Compared to KH-CB19, CLK-IN-T3 exhibits approximately two orders of magnitude higher potency and selectivity for CLK subtypes. CLK-IN-T3 regulates RNA splicing by dose-dependently promoting exon skipping and intron retention. Surprisingly, it induces co-gene transcription by influencing 3' end processing and splicing factor activity. Based on previous studies of other CLK inhibitors [1], this compound may have therapeutic potential for cancers with dysregulated CLK activity, such as breast cancer (CLK2) and renal cell carcinoma (CLK1).
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| Molecular Formula |
C28H30N6O2
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|---|---|
| Molecular Weight |
482.576805591583
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| Exact Mass |
482.243
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| Elemental Analysis |
C, 64.79; H, 6.02; Cl, 6.83; N, 16.19; O, 6.16
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| CAS # |
2109805-56-1
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| Related CAS # |
2109805-56-1;
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| PubChem CID |
132585205
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.659
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| LogP |
2.36
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
36
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| Complexity |
768
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C(C)(C)C1C=CC(C(NC2=CN3C=C(C4C=CN=CC=4)C=CC3=N2)=O)=CC=1)N1CCN(C)CC1
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| InChi Key |
IEFFSHLHNYVSEF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H30N6O2/c1-28(2,27(36)33-16-14-32(3)15-17-33)23-7-4-21(5-8-23)26(35)31-24-19-34-18-22(6-9-25(34)30-24)20-10-12-29-13-11-20/h4-13,18-19H,14-17H2,1-3H3,(H,31,35)
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| Chemical Name |
4-[2-methyl-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl]-N-(6-pyridin-4-ylimidazo[1,2-a]pyridin-2-yl)benzamide
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| Synonyms |
T3; CLK Inhibitor T3; T3-CLK; MDK 5561; MDK-5561; MDK5561; MDK-5561 hydrochloride; MDK-5561 HCl
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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: ~4.8 mg/mL (~10.0 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.31 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 (4.31 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0722 mL | 10.3610 mL | 20.7220 mL | |
| 5 mM | 0.4144 mL | 2.0722 mL | 4.1444 mL | |
| 10 mM | 0.2072 mL | 1.0361 mL | 2.0722 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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