GW296115
Based on 1 Customer Validation
GW296115 is a multi-target inhibitor with the following IC50 values against its targets: 8.4 nM for BRSK2, 21 nM for BRSK1, 1.8 μM for PDGFRβ, 5.5 nM for STK17B/DRAK2, 28 nM for DRAK1, 20 nM for PHKG1, and 89 nM for DCAMKL3. GW296115 downregulates the phosphorylation of S317 site on ULK1 and S351 site on P62, which are AMPK substrates driven by BRSK2. GW296115 does not alter the phosphorylation level of AMPK at T172, reduces nutrient deprivation-mediated Autophagy and autophagosome formation, and enhances Apoptosis. GW296115 exhibits anticancer activity against triple-negative breast cancer. GW296115 is applicable for breast cancer-related research.
For research use only. We do not sell to patients.
- Purity : 98.68%
- CAS No.: 118458-58-5
- Formula: C22H15N3O4
- Molecular Weight:385.37
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Storage:
4°C, sealed storage, away from moisture
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Biological Activity
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Chemical Information
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Solvent & Solubility
- Protocols
- Purity & Documentation
- References
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Help & FAQs
Biological Activity
Description
IC50 & Target
[1]|
PDGFRβ 1.8 μM (IC50) |
ULK1 |
DRAK1 28 nM (IC50) |
DRAK2 5.5 nM (IC50) |
Cellular Effect
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Cell Line
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Type | Value | Description | References |
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| A10 | IC50 |
15 nM
Compound: 3744W
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Inhibition of PDGF-dependent PDGFR autophosphorylation in rat A10 cells
Inhibition of PDGF-dependent PDGFR autophosphorylation in rat A10 cells
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[PMID: 10048786] |
| A10 | IC50 |
10 nM
Compound: 3744W
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Inhibition of PDGF-dependent PDGFR-mediated cell proliferation in rat A10 cells
Inhibition of PDGF-dependent PDGFR-mediated cell proliferation in rat A10 cells
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[PMID: 10048786] |
In Vitro
GW296115 (1 μM) inhibits 25 out of 403 wild-type human kinases by over 90% at a concentration of 1 μM, with a selectivity index of 0.062[1].
GW296115 potently inhibits 6 IDG dark kinases in enzymatic assays, with IC50 values ranging from 5.5 nM (DRAK2) to 89 nM (DCAMKL3)[1].
GW296115 (1 μM; 72 h) exerts no effect on cell proliferation in 17 tested cancer cell lines and normal cell lines (including non-malignant MCF10A and MRC-5) following treatment at 1 μM for 72 h[1].
GW296115 (incubated for 2 h) potently binds to BRSK2 in living HEK293 cells with an IC50 of 107 nM, confirming that it is a cell-active BRSK2 inhibitor[1].
GW296115 (2.5 μM; 2-6 h) abolishes BRSK2-induced phosphorylation of AMPK substrates in HEK293T cells (this effect is observed at both 2 h and 6 h) and induces hyperphosphorylation of BRSK2 at the T174 site[1].
GW296115 (2-5 μM) inhibits autophagy and enhances apoptosis in MDA-MB-231 triple-negative breast cancer cells, while it also inhibits autophagy and enhances apoptosis in BT-474 estrogen receptor-positive breast cancer cells[2].
GW296115 (2 μM; 24 h) inhibits the growth of MDA-MB-231 triple-negative breast cancer cells[2].
GW296115 (2-3 μM; 72 h) reduces the 3D invasiveness of MDA-MB-231 triple-negative breast cancer cells[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.
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Cell Line:17 cancer and normal cell lines (including SUM159, MCF7, MCF10A, HCC1954, COV362, Kuramochi, PC-3, DU145, H729, HCT116, A549, MRC-5, Colo829, A375, Cas1, HPAF-II, Panc-1)
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Concentration:1 μM
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Incubation Time:72 h
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Result:Did not impact cell growth in any of the tested cell lines, and is considered generally non-toxic to cells.
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Cell Line:HEK293T cells transiently expressing hcRED, wild-type BRSK2, or kinase-dead BRSK2 (K48A, T174A)
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Concentration:2.5 μM
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Incubation Time:2 h, 6 h
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Result:Ablated wild-type BRSK2-induced AMPK substrate phosphorylation at both 2-h and 6-h time points.
Left phosphorylation of AMPK at T172 unaltered, but hyper-induced phosphorylation of BRSK2 at T174 in all samples except those expressing BRSK2 T174A.
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Cell Line:MDA-MB-231
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Concentration:2-3 μM
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Incubation Time:72 h
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Result:Reduced 3D Matrigel tumor cell invasiveness (invadopodia per spheroid) by over 75% compared to vehicle control.
Chemical Information
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CAS No. 118458-58-5
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Appearance Solid
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Molecular Weight 385.37
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Formula C22H15N3O4
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Color Yellow to brown
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SMILES
O=C1NC(C2=C3C4=C(C=CC(OC)=C4)NC3=C5NC6=CC=C(OC)C=C6C5=C21)=O
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
4°C, sealed storage, away from moisture
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
Solvent & Solubility
In Vitro:
DMSO : 50 mg/mL (129.75 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
In Vivo:
Select the appropriate dissolution method based on your experimental animal and administration route.
- For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
- To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for In Vivo experiments, it is recommended to prepare freshly and use it on the same day.
- The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: ≥ 2.5 mg/mL (6.49 mM); Clear solution
This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: ≥ 2.5 mg/mL (6.49 mM); Suspended solution
This protocol yields a suspended solution of ≥ 2.5 mg/mL (saturation unknown). Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:
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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
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%DMSO +
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
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%+
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+%Tween-80 + +
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%Saline +
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Working solution concentration: 0.22 mg/mL
Method for preparing stock solution: mg drug dissolved in μL DMSO. Stock solution concentration: mg/mL. * In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)
1. Take μL DMSO stock solution;
2. Add μL .
μL , mix evenly;
3. Then add μL Tween 80, mix evenly;
4. Then add μL
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Protocols
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Kinase activity and phosphorylation assays
Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of 32P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.
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Western Blot
Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.
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Apoptosis
Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics.
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TUNEL staining for apoptotic DNA fragmentation
TUNEL staining detects DNA strand breaks by using terminal deoxynucleotidyl transferase to add labeled nucleotides to exposed 3′-OH DNA termini, generating either microscopic staining in fixed cells or tissue sections, or fluorescence/cytometric signal in cell suspensions. TUNEL positivity reflects DNA fragmentation but should not be interpreted alone as definitive apoptosis, because TUNEL can also label necrotic, autolytic, mechanically damaged, or DNA-repair-associated DNA breaks.
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Cell Cytotoxicity Assay
Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.
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Annexin V plus membrane-impermeant dye apoptosis staining
Annexin V-based apoptosis assays rely on the detection of phosphatidylserine (PS) externalization from the inner leaflet of the plasma membrane to the outer leaflet, an early biochemical hallmark of apoptosis. Fluorescently labeled Annexin V binds PS in a calcium-dependent manner, enabling identification of early apoptotic cells by flow cytometry or fluorescence microscopy. When combined with a membrane-impermeant DNA-binding dye (e. g. , propidium iodide), this approach allows discrimination between viable (Annexin V−/dye−), early apoptotic (Annexin V+/dye−), and late apoptotic or necrotic (Annexin V+/dye+) cell populations by assessing membrane integrity and PS exposure.
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Autophagy
Autophagy is a process in which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy related gene (Atg). Microtubule-associated proteins light chain 3 (LC3) is recognized as autophagy marker, which transfers from cytoplasmic LC3 (LC3-I) to membrane type (LC3-II). LC3-II/I ratio could be detected by Western Blot and fluorescence microscopy.
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Apoptosis Solutions
Apoptosis is a regulated, generally non-lytic cell-death pathway that removes unwanted, damaged, infected, or abnormal cells through coordinated morphological changes, caspase activation, DNA fragmentation, and membrane remodeling. The intrinsic apoptosis pathway is controlled mainly by mitochondrial outer membrane permeabilization, BCL-2 family proteins, cytochrome c release, apoptosome formation, caspase-9 activation, and downstream executioner caspase-3/7 activation. The extrinsic apoptosis pathway is initiated by death receptors such as Fas, TNFR, and TRAIL receptors, which recruit adaptor proteins and activate caspase-8 before engaging executioner caspases or mitochondrial amplification through BID cleavage. Apoptosis is linked to many phenotypes, including cancer cell killing, tissue homeostasis, immune regulation, neurodegeneration, infection response, and treatment-induced cytotoxicity; unresolved questions include how apoptosis interacts with necroptosis, pyroptosis, ferroptos
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Protocol for Kinase activity and phosphorylation assays
Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.
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Lysosome and acidic-vesicle live-cell staining
Lysosome and acidic-vesicle live-cell staining detects acidic intracellular compartments by using membrane-permeant acidotropic probes that accumulate in low-pH vesicles, including lysosomes, late endosomes, autolysosomes, and acidic phagosomes. LysoTracker staining is commonly used as an intensity-based readout of acidic lysosomal compartment abundance or enlargement, while acridine orange produces green fluorescence in less concentrated compartments and red fluorescence after concentration-dependent accumulation in acidic vesicular organelles. Loss or reduction of acridine-orange red signal can be used as a readout of lysosomal membrane permeabilization or reduced acidic-vesicle integrity. This protocol is designed for live cultured cells and can be adapted for fluorescence microscopy, high-content imaging, plate-reader readout, or flow cytometry when the selected literature supports the readout. Because these dyes report acidotropic accumulation rather than lysosome identity alone,
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Macroautophagy Solutions
Macroautophagy is a conserved lysosome-dependent degradation pathway in which cytoplasmic material is sequestered into double-membrane autophagosomes and delivered to lysosomes for degradation and recycling. The pathway supports cellular homeostasis during nutrient limitation, organelle stress, protein-aggregate accumulation, infection, differentiation, and tissue remodeling by coupling cargo sequestration, autophagosome maturation, lysosomal fusion, and degradation of cargo-derived macromolecules. The core molecular sequence includes initiation by nutrient- and stress-regulated autophagy machinery, autophagosome nucleation, LC3/ATG8-family conjugation to autophagosomal membranes, cargo selection through receptors such as SQSTM1/p62, autophagosome-lysosome fusion, and lysosomal degradation. LC3 was identified as a mammalian homolog of yeast Atg8 that localizes to autophagosomal membranes after processing, and p62/SQSTM1 was shown to connect ubiquitinated cargo with autophagic degradati
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Breast Cancer Modeling
Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.
Purity & Documentation
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Data Sheet (282 KB)
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SDS (252 KB)
- English - EN (252 KB)
- Français - FR (252 KB)
- Deutsch - DE (252 KB)
- Norwegian - NO (252 KB)
- Español - ES (252 KB)
- Swedish - SV (252 KB)
- Italian - IT (252 KB)
- Korean - KR (252 KB)
- Portuguese - PT (252 KB)
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Handling Instructions (2659 KB)
References
[1]. Tamir TY, et al. PKIS deep dive yields a chemical starting point for dark kinases and a cell active BRSK2 inhibitor. Sci Rep. 2020 Sep 28;10(1):15826. [Content Brief]
[2]. Maiti A, et al. BRSK2 plays a role in autophagy and cancer cell growth and survival under nutrient deprivation stress via the PIK3C3 pathway. Sci Rep. 2025 Nov 19;15(1):40651. [Content Brief]
Complete Stock Solution Preparation Table
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
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| DMSO | 1 mM | 2.5949 mL | 12.9745 mL | 25.9491 mL | 64.8727 mL |
| 5 mM | 0.5190 mL | 2.5949 mL | 5.1898 mL | 12.9745 mL | |
| 10 mM | 0.2595 mL | 1.2975 mL | 2.5949 mL | 6.4873 mL | |
| 15 mM | 0.1730 mL | 0.8650 mL | 1.7299 mL | 4.3248 mL | |
| 20 mM | 0.1297 mL | 0.6487 mL | 1.2975 mL | 3.2436 mL | |
| 25 mM | 0.1038 mL | 0.5190 mL | 1.0380 mL | 2.5949 mL | |
| 30 mM | 0.0865 mL | 0.4325 mL | 0.8650 mL | 2.1624 mL | |
| 40 mM | 0.0649 mL | 0.3244 mL | 0.6487 mL | 1.6218 mL | |
| 50 mM | 0.0519 mL | 0.2595 mL | 0.5190 mL | 1.2975 mL | |
| 60 mM | 0.0432 mL | 0.2162 mL | 0.4325 mL | 1.0812 mL | |
| 80 mM | 0.0324 mL | 0.1622 mL | 0.3244 mL | 0.8109 mL | |
| 100 mM | 0.0259 mL | 0.1297 mL | 0.2595 mL | 0.6487 mL |
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