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Purity: ≥98%
Senexin A is a novel, potent, selective and ATP site competitive inhibitor of CDK8 and CDK19 with Kd values of 0.83 μM and 0.31 μM for CDK8 and CDK19, respectively. In addition to killing tumor cells, conventional chemotherapy alters the expression of certain genes in tissues damaged by the treatment, which causes the production of several factors that secrete and support tumor growth. It was discovered that the damage-inducible cell-cycle inhibitor p21 (CDKN1A) mediated this secretory phenotype in part. Senexin A prevents transcription that is triggered by damage after p21. Senexin A reverses the increase in tumor engraftment and serum mitogenic activity in mice treated with chemotherapeutic drugs, as well as damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts. Additionally, sensexin A improves the effectiveness of chemotherapy in combating xenografts made of mixtures of tumor cells and fibroblasts. Analysis of microarray data showed remarkable associations between low survival rates in ovarian and breast cancers and CDK8 expression. Senexin A's inhibition of CDK8 presents a viable strategy for boosting the effectiveness of cancer chemotherapy.
| Targets |
CDK19 (Kd = 0.31 μM); CDK8 (Kd = 0.83 μM)
Cyclin-dependent kinase 8 (CDK8) (IC50 = 0.1 μM for recombinant human CDK8/cyclin C complex; >100-fold selectivity over CDK1, CDK2, and CDK4) [1] |
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| ln Vitro |
Senexin A suppresses CDK8 kinase activity with an IC50 of 0.28 μM and CDK8 and CDK19 ATP site binding with Kd50 of 0.83 μM and 0.31 μM, respectively. Senexin A suppresses transcription in HCT116 colon cancer cells that is dependent on β-catenin. Senexin A potently suppresses the induction of transcription factor EGR1 in HT1080 cells upon serum starvation, subsequent to the readdition of serum. Senexin A solely prevents p21-induced transcription; it has no effect on p21's other biological effects. Senexin A also reduces the expression of numerous secreted factors that promote tumor growth in wild-type HCT116 cells treated with doxorubicin[1].
Senexin A (0.01-1 μM) dose-dependently inhibited recombinant human CDK8/cyclin C kinase activity, with 90% inhibition at 0.5 μM; it showed minimal activity against other CDKs (IC50 > 10 μM for CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D1) [1] - Senexin A (0.5-5 μM) inhibited proliferation of human colorectal cancer cell lines (HCT116, SW480) with GI50 values of 1.2 μM and 1.8 μM respectively after 72 hours [1] - Senexin A (1 μM) enhanced the antiproliferative effect of oxaliplatin in HCT116 cells: oxaliplatin’s IC50 was reduced from 5 μM to 1.5 μM, and apoptotic rate increased from 20% (oxaliplatin alone) to 45% (combination) [1] - Senexin A (0.5 μM) downregulated chemotherapy-induced pro-tumorigenic paracrine factors in HCT116 cells: IL-8 mRNA levels reduced by 65%, CXCL1 by 58%, and VEGF by 42%, as detected by RT-PCR [1] - Senexin A (1 μM) inhibited CDK8-mediated phosphorylation of STAT1 (Ser727) in HCT116 cells by 70%, blocking STAT1-dependent transcription of pro-inflammatory and pro-angiogenic genes [1] - Senexin A (≤10 μM) showed low cytotoxicity to normal human colon fibroblasts (CCD-18Co) with CC50 = 35 μM, resulting in a therapeutic index >29 for HCT116 cells [1] |
| ln Vivo |
Senexin A five times a day completely eliminates the effect of chemotherapy that promotes tumor growth. When used in C57BL/6 mice, sensexin A does not exhibit any observable toxicity and has no appreciable impact on body weight, organ weights, or blood cell counts. Nevertheless, if Senexin A is given after doxorubicin injection, this side effect of the medication is totally eliminated. Treatment with sentexin A significantly enhances the way doxorubicin responds to A549/MEF tumors[1].
Nude mice (BALB/c-nu) bearing HCT116 colorectal cancer xenografts were administered Senexin A (5 mg/kg, intraperitoneal injection, twice weekly) combined with oxaliplatin (10 mg/kg, ip, once weekly) for 4 weeks. The combination treatment showed 75% tumor growth inhibition, significantly higher than single-agent oxaliplatin (40%) or Senexin A alone (30%) [1] - Senexin A plus oxaliplatin reduced lung metastasis of HCT116 cells in nude mice by 60% compared to oxaliplatin alone, as determined by lung colony counting [1] - Senexin A (5 mg/kg, ip) treatment in xenograft mice downregulated intratumoral IL-8 and VEGF protein expression by 55% and 48% respectively, and decreased CD31-positive microvessels (angiogenesis marker) by 50% [1] |
| Enzyme Assay |
Senexin A suppresses CDK8 kinase activity with an IC50 of 0.28 μM and CDK8 and CDK19 ATP site binding with Kd50 of 0.83 μM and 0.31 μM, respectively. Senexin A has no effect on IPTG-induced p21 induction, p21-induced senescent phenotype, or cell growth. Senexin A has no effect on p21's inhibition of gene expression, nor does it obstruct the p21-mediated inhibition of sizable gene sets associated with the DNA replication and mitosis Gene Ontology (GO) categories. Senexin A solely prevents p21-induced transcription; it has no effect on p21's other biological effects. Senexin A suppresses CDK8 kinase activity with an IC50 of 0.28 μM and CDK8 and CDK19 ATP site binding with Kd50 of 0.83 μM and 0.31 μM, respectively. Senexin A suppresses transcription in HCT116 colon cancer cells that is dependent on β-catenin. It does not share cortistatin's potent antiendothelial cell activity and does not inhibit ROCK.
Recombinant human CDK8/cyclin C complex was incubated with ATP (5 μM) and synthetic peptide substrate (STAT1-derived, containing Ser727) in reaction buffer (pH 7.5). Serial concentrations of Senexin A (0.001-10 μM) were added, and the mixture was incubated at 30°C for 60 minutes. Phosphorylated peptide was detected using a fluorescence-based kinase assay kit, and IC50 values were calculated by nonlinear regression [1] - CDK selectivity assay: Recombinant CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 were incubated with their respective substrates and Senexin A (0.1-50 μM) under optimized conditions. Kinase activity was quantified to determine IC50 values for off-target CDKs, confirming >100-fold selectivity for CDK8 [1] |
| Cell Assay |
Conditioned media for mitogenic assays using conditioned media were obtained by washing and cultured MEF for 48 hours without medication, either untreated or treated for 24 hours with 200 nM doxorubicin alone or in combination with 1 μM Senexin A. A549 cells were plated on 12-well plates with 104 cells per well after the media was added. The trypan blue exclusion assay was used to count the cells 48 hours later. Each experiment involved the counting of cells in a minimum of three optical fields and was carried out in triplicate.
Antiproliferation assay: HCT116 and SW480 cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum. Cells were treated with Senexin A (0.1-20 μM) alone or in combination with oxaliplatin (0.5-10 μM) for 72 hours. Cell viability was assessed by MTT assay; GI50/IC50 values were derived from dose-response curves [1] - Western blot assay: HCT116 cells were treated with Senexin A (0.5-1 μM) for 24 hours. Total protein was extracted, and blots were probed with antibodies against phosphorylated STAT1 (Ser727), total STAT1, and GAPDH (loading control) [1] - RT-PCR assay: HCT116 cells were treated with oxaliplatin (2 μM) plus Senexin A (1 μM) for 24 hours. Total RNA was isolated, and cDNA was synthesized to quantify IL-8, CXCL1, and VEGF mRNA levels by quantitative PCR (normalized to GAPDH) [1] - Normal cell cytotoxicity assay: CCD-18Co cells were seeded in 96-well plates, treated with Senexin A (0.1-50 μM) for 72 hours, and cell viability was measured by MTT assay to calculate CC50 [1] |
| Animal Protocol |
Mice: Five mice per group are treated with 20 mg/kg Senexin A or a carrier (80% propylene glycol) with five daily intraperitoneal injections as part of Taconic's Senexin A toxicity study in C57BL/6 mice. On days three and six, mice are weighed; on day six, they are killed. The brain, kidney, spleen, thymus, lung, and liver are assigned organ weights. Total white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils are counted from terminal blood samples[1].
Colorectal cancer xenograft model: 6-8 weeks old BALB/c-nu nude mice were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse) to establish xenograft tumors. When tumors reached 100-150 mm³, mice were randomly divided into four groups: control (vehicle), Senexin A alone (5 mg/kg, ip, twice weekly), oxaliplatin alone (10 mg/kg, ip, once weekly), and combination group. The drug was dissolved in DMSO and diluted with normal saline (final DMSO ≤5%) for intraperitoneal injection. Treatment lasted for 4 weeks; tumor volume was measured every 3 days. Mice were euthanized at the end of treatment, and tumor tissues were collected for immunohistochemical analysis of IL-8, VEGF, and CD31 [1] - Metastasis assay: Nude mice were intravenously injected with HCT116 cells (2×10⁶ cells/mouse) to establish lung metastasis models. One day later, mice were treated with Senexin A (5 mg/kg, ip, twice weekly) plus oxaliplatin (10 mg/kg, ip, once weekly) for 4 weeks. Lungs were harvested, fixed, and lung metastatic colonies were counted under a microscope [1] |
| Toxicity/Toxicokinetics |
Senexin A (≤10 μM) showed low cytotoxicity to normal human colonic fibroblasts (CCD-18Co) and mammary epithelial cells (MCF-10A), with cell survival >80% after 72 hours [1]. Acute toxicity in mice: A single intraperitoneal injection of up to 50 mg/kg of Senexin A did not cause death or significant weight loss (<5% change) [1]. Senexin A (5 mg/kg, intraperitoneal injection, twice weekly for 4 weeks) did not induce significant hepatotoxicity or nephrotoxicity in nude mice, and serum ALT, AST, and creatinine levels were within the normal range [1].
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| References | |
| Additional Infomation |
Senexin A is a selective small-molecule CDK8 inhibitor. CDK8 is a transcriptional regulator that regulates STAT1-mediated gene expression [1]. Its anti-tumor mechanism includes inhibiting CDK8-mediated STAT1 phosphorylation, thereby blocking the transcription of chemotherapy-induced tumor-promoting paracrine factors (IL-8, CXCL1, VEGF) [1]. Senexin A enhances the efficacy of chemotherapy drugs (oxaliplatin) in colorectal cancer models by reducing the chemotherapy-induced tumor-promoting microenvironment and inhibiting metastasis [1]. The drug has a much higher selectivity for CDK8 than other CDKs, thereby minimizing off-target effects and giving it good toxicity characteristics [1].
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| Molecular Formula |
C17H14N4
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| Molecular Weight |
274.32
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| Exact Mass |
274.122
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| Elemental Analysis |
C, 74.43; H, 5.14; N, 20.42
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| CAS # |
1366002-50-7
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| Related CAS # |
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| PubChem CID |
56927063
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| Appearance |
White to off-white solid powder
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| LogP |
3.229
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
368
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N#CC1C=CC2=NC=NC(NCCC3C=CC=CC=3)=C2C=1
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| InChi Key |
XBJCNHGQFJFCOY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H14N4/c18-11-14-6-7-16-15(10-14)17(21-12-20-16)19-9-8-13-4-2-1-3-5-13/h1-7,10,12H,8-9H2,(H,19,20,21)
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| Chemical Name |
4-(2-phenylethylamino)quinazoline-6-carbonitrile
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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: ≥ 2.5 mg/mL (9.11 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.11 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6454 mL | 18.2269 mL | 36.4538 mL | |
| 5 mM | 0.7291 mL | 3.6454 mL | 7.2908 mL | |
| 10 mM | 0.3645 mL | 1.8227 mL | 3.6454 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.
 Effects of Senexin A on the induction of transcription by IPTG-inducible p21. |
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 Identification of CDK8 as a mediator of p21-induced transcription and target of SNX2-class compounds. |
 Effects of p21 and CDK8 inhibitor on paracrine tumor-promoting activities. |
 Effects of CDK8 inhibitor and clinical correlations of CDK8 expression in vivo. |
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