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Purity: ≥98%
STK16-IN-1 is a novel and highly selective ATP competitive inhibitor of STK16 kinase with an IC50 of 295 nM. STK16 is a serine/threonine protein kinase that is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. STK16-IN-1 exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16.
| Targets |
STK16 (serine/threonine protein kinase) (IC₅₀ = 0.295 μM for inhibitory activity against STK16 kinase; S score (1) = 0.0 in KinomeScan profiling assay, indicating excellent selectivity across the kinome) [1]
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| ln Vitro |
As determined by the KinomeScanTM profiling experiment, STK16-IN-1 has strong inhibitory action against STK16 kinase (IC50=0.295 μM) and good selectivity throughout the kinome. The inhibitory concentration of STK16-IN-1 on mTOR kinase is 5.56 μM. Treatment of MCF-7 cells with STK16-IN-1 causes a decrease in the number of cells and a build-up of binucleated cells; this effect can be replicated by RNA interference (RNAi) knockdown of STK16. The anti-proliferative effects of chemotherapeutics such cisplatin, doxorubicin, colchicine, and paclitaxel are slightly enhanced when STK16-IN-1 is co-treated with these drugs. A helpful tool compound for better understanding the biological roles of STK16) is STK16-IN-1[1].
1. STK16-IN-1 is a highly selective ATP-competitive inhibitor of STK16 kinase, with an IC₅₀ of 0.295 μM for STK16 kinase inhibition. KinomeScan profiling assay confirmed its excellent selectivity across the kinome (S score (1) = 0.0). In MCF-7 breast cancer cells, treatment with STK16-IN-1 led to a reduction in cell number and accumulation of binucleated cells; this cellular phenotype was consistent with the effect of STK16 RNAi knockdown in MCF-7 cells. Co-treatment of MCF-7 cells with STK16-IN-1 and chemotherapeutic drugs (cisplatin, doxorubicin, colchicine, paclitaxel) slightly potentiated the antiproliferative effects of these chemotherapeutics [1] |
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| ln Vivo |
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| Enzyme Assay |
STK16-IN-1 is a novel and highly selective ATP competitive inhibitor of STK16 kinase with an IC50 of 295 nM. STK16 is a serine/threonine protein kinase that is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. STK16-IN-1 exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). STK16-IN-1 is generally prepared with 1:3 serial dilutions for 4 concentrations (100 nM, 50 nM, 20 nM, and 10 nM); 6 concentrations are used (1 mM to 10 μM) for ATP competition experiments. The kinase reaction is performed with 1×kinase reaction buffer. Reactions in each well are started immediately by adding ATP and kept going for half an hour under 37°C. After the plate cooled for 5 minutes at room temperature, 5 μL of ADP-Glo reagent is added into each well to stop the reaction and consume the remaining ADP within 40 minutes. At the end, 10 μL of kinase detection reagent is added into the well and incubated for 1 hour to produce a luminescence signal.
1. STK16 kinase activity inhibition assay: Recombinant STK16 kinase protein was incubated with different concentrations of STK16-IN-1 in a reaction buffer containing ATP and a specific peptide substrate of STK16. The reaction mixture was incubated at an appropriate temperature for a set period to allow kinase-mediated phosphorylation of the substrate. The amount of phosphorylated substrate was detected using a luminescent or colorimetric method to quantify STK16 kinase activity. The IC₅₀ value (0.295 μM) for STK16-IN-1 against STK16 kinase was calculated by fitting the dose-response curve of inhibitory activity. Kinome selectivity assay (KinomeScan profiling): A panel of hundreds of human kinases was screened to evaluate the inhibitory activity of STK16-IN-1 against each kinase at a fixed concentration. The binding affinity of STK16-IN-1 to each kinase was measured, and the S score (1) was calculated to assess the selectivity; an S score (1) of 0.0 indicated that STK16-IN-1 had no significant inhibitory effect on other kinases except STK16 [1] |
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| Cell Assay |
In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16. MCF-7, HCT116, HeLa cells are treated with STK16-IN-1 (0, 5, 10 μM) for 72 hours and apoptotic cells are analyzed by flow cytometry using Annexin V/PI apoptosis detection kit.
1. MCF-7 cell proliferation and phenotype assay: MCF-7 cells were seeded in 96-well or 6-well culture plates and cultured to reach logarithmic growth phase. Cells were treated with different concentrations of STK16-IN-1 (or vehicle control) for a specified time. For cell number quantification: Cells were trypsinized, and the number of viable cells was counted using a hemocytometer or an automated cell counter. For binucleated cell analysis: Cells were fixed with paraformaldehyde, stained with DAPI to label nuclei, and observed under a fluorescence microscope; the percentage of binucleated cells was counted. STK16 RNAi knockdown assay: MCF-7 cells were transfected with STK16-specific siRNA or negative control siRNA using a transfection reagent. After transfection for a certain time, cell number and nuclear morphology (binucleated cells) were analyzed as described above to compare with the effects of STK16-IN-1 treatment. Combination treatment with chemotherapeutics: MCF-7 cells were treated with STK16-IN-1 in combination with cisplatin, doxorubicin, colchicine, or paclitaxel at various concentrations. After incubation for a set period, cell viability was assessed using MTT or CCK-8 assay to determine the antiproliferative effect of the combination treatment and compare it with single-agent treatment [1] |
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| Animal Protocol |
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| References | |||
| Additional Infomation |
1. STK16 is a serine/threonine protein kinase that is universally expressed in all eukaryotes and is involved in a variety of cellular processes, such as VEGF and cargo secretion, but its underlying mechanisms remain unclear. STK16-IN-1 was discovered by screening a specific library of kinase inhibitors and is an ATP-competitive inhibitor of STK16. Its high selectivity and potent inhibitory activity make it a useful tool compound for elucidating the biological functions of STK16 [1]
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| Molecular Formula |
C17H12FN3O
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| Molecular Weight |
293.3
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| Exact Mass |
293.096
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| Elemental Analysis |
C, 69.62; H, 4.12; F, 6.48; N, 14.33; O, 5.45
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| CAS # |
1223001-53-3
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| Related CAS # |
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| PubChem CID |
58525066
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| Appearance |
Solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
552.4±50.0 °C at 760 mmHg
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| Flash Point |
287.9±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.713
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| LogP |
3.41
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
22
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| Complexity |
483
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1=CC=C(N2C(C(C=CC2=O)=CN3)=C4C3=NC=C4)C=C1C
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| InChi Key |
WQNRDXHKVSKUPI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H12FN3O/c1-10-8-12(3-4-14(10)18)21-15(22)5-2-11-9-20-17-13(16(11)21)6-7-19-17/h2-9H,1H3,(H,19,20)
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| Chemical Name |
1-(4-fluoro-3-methylphenyl)-7H-pyrrolo[2,3-h][1,6]naphthyridin-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) |
DMSO: ~59 mg/mL ( 201.15 mM)
Water: <4 mg/mL Ethanol: Insoluble |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.52 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 (8.52 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 | 3.4095 mL | 17.0474 mL | 34.0948 mL | |
| 5 mM | 0.6819 mL | 3.4095 mL | 6.8190 mL | |
| 10 mM | 0.3409 mL | 1.7047 mL | 3.4095 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.
ACS Chem Biol.2016 Jun 17;11(6):1537-43. |
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