WNK2-IN-1
WNK2-IN-1 is a WNK2 inhibitor. WNK2-IN-1 inhibits the kinase activity of recombinant WNK2 in a dose-dependent manner, and simultaneously partially inhibits WNK3 and WNK4. WNK2-IN-1 modulates WNK2-SPAK downstream signaling by inhibiting WNK2-mediated SPAK phosphorylation, and further suppresses the proinflammatory transcriptional response induced by IL1β. WNK2-IN-1 can be used in studies related to the WNK2 signaling pathway and osteoarthritis.
For research use only. We do not sell to patients.
- CAS No.: 2728520-90-7
- Formula: C19H20F6N4O
- Molecular Weight:434.38
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Storage:
Please store the product under the recommended conditions in the Certificate of Analysis.
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Biological Activity
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Chemical Information
- Protocols
- Purity & Documentation
- References
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Help & FAQs
Biological Activity
Description
IC50 & Target
[1]|
WNK2 |
WNK3 |
WNK4 |
In Vitro
WNK2-IN-1 (compound M04) (0.1-1000 μM; 24 h) exhibits an IC50 of 416 μM for cell viability in human T/C-28a2 chondrocytes[1].
WNK2-IN-1 (1-400 μM) reduces pSPAK levels in T/C-28a2 chondrocytes in a dose-dependent manner; at 200-400 μM, its reducing effect on pSPAK is comparable to that of the pan-WNK inhibitor WNK463 (HY-100626)[1].
WNK2-IN-1 (1-200 μM) reduces WNK2 kinase activity in a dose-dependent manner in recombinant kinase assays; it does not affect WNK1 under the same conditions, and decreases WNK3 and WNK4 activity in a dose-dependent manner. However, the degree of inhibition of WNK3/WNK4 is weaker than that of WNK2, and does not reach the inhibitory level of WNK463[1].
WNK2-IN-1 (400 μM; 5 min) reduces pSPAK to levels comparable to those achieved with WNK463 in T/C-28a2 chondrocytes; (R)-WNK2-IN-1 produces a similar reduction in pSPAK, whereas (S)-WNK2-IN-1 exhibits only a minor effect, indicating that the R-enantiomer is the active form[1].
Compared with WNK463 (10 μM), WNK2-IN-1 (100 μM; 24 h) upregulates MMP2 and MMP13 and downregulates GREM2 and BMP4 in primary human chondrocytes not stimulated with IL1β; meanwhile, it enhances cell growth, Hedgehog signaling pathway inhibition, and immune response-related pathways, and downregulates autophagy-, starvation response-, and chemical stress-related pathways[1].
WNK2-IN-1 (100 μM; 24 h; 10 ng/mL IL1β; 100 mM Sorbitol (HY-B0400)) reduces the expression of IL6 and CCL2 to control levels, increases COL2A1 expression, and prevents IL1β-induced downregulation of ACAN in T/C-28a2 chondrocytes[1].
WNK2-IN-1 (100 μM; 24 h; 10 ng/mL IL1β; 100 mM Sorbitol) reduces the expression of CCL2 and FGF1 in primary human chondrocytes; no significant changes in gene expression are observed in control chondrocytes not stimulated with IL1β[1].
Compared with WNK463 (10 μM), WNK2-IN-1 (100 μM; 24 h; 10 ng/mL IL1β) reduces the expression of BMP2, BMP4, NFKB1, MMP13 and FN1 and upregulates SLC1A5 and GREM1; meanwhile, it enhances pathways related to growth, TGFβ signaling, circadian rhythm and potassium transport, and downregulates pathways associated with TLR, interferon α/β/γ, interleukin, and RIPK-mediated NFκB activation[1].
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:human T/C-28a2 chondrocytes
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Concentration:0.1-1000 μM
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Incubation Time:24 h (before adding XTT); 4 h (additional incubation with XTT working solution)
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Result:Exhibited very low cytotoxicity in human T/C-28a2 chondrocytes, with a cell viability IC50 value of 416 μM.
Showed an IC50 value substantially higher than the cytotoxic IC50 values of other screened candidate WNK2 inhibitors, indicating excellent tolerability.
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Cell Line:human T/C-28a2 chondrocytes
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Concentration:1, 10, 50, 100, 200, 400 μM
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Incubation Time:15 min (pre-exposure before media replacement); 10 min (further incubation under isotonic or hyperosmotic 500 mOsm conditions prior to cell harvest)
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Result:Produced a dose-dependent reduction in pSPAK levels under both isotonic (300 mOsm) and acute 500 mOsm osmotic stress conditions.
Reduced pSPAK accumulation to levels comparable to the pan-WNK control inhibitor at 200 μM and 400 μM, confirming effective inhibition of WNK2-mediated SPAK phosphorylation.
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Cell Line:human T/C-28a2 chondrocytes
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Concentration:400 μM (racemic WNK2-IN-1 (M04)); 400 μM ((R)-M04); 400 μM ((S)-M04)
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Incubation Time:5 min (incubation under 500 mOsm osmotic stress)
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Result:Reduced pSPAK levels to an extent comparable to the control pan-WNK inhibitor when using 400 μM racemic WNK2-IN-1 (M04) or 400 μM (R)-M04.
Produced only a minor inhibitory effect on pSPAK accumulation when using 400 μM (S)-M04.
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Cell Line:human T/C-28a2 chondrocytes
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Concentration:100 μM
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Incubation Time:24 h (during 10 ng/mL IL1β stimulation in 100 mM Sorbitol hyperosmotic media)
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Result:Reduced the elevated expression of proinflammatory genes IL6 and CCL2 back to near non-stimulated control levels in IL1β-stimulated T/C-28a2 chondrocytes.
Increased the expression of the cartilage anabolic collagen gene COL2A1, and prevented the IL1β-induced downregulation of the key cartilage proteoglycan gene ACAN.
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Cell Line:primary human articular chondrocytes
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Concentration:100 μM
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Incubation Time:24 h (during 10 ng/mL IL1β stimulation in 100 mM Sorbitol hyperosmotic media)
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Result:Significantly reduced the IL1β-induced upregulation of the proinflammatory genes CCL2 and FGF1 in IL1β-stimulated primary human chondrocytes.
Did not induce any significant changes in baseline gene expression in non-IL1β-stimulated control chondrocytes.
Chemical Information
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CAS No. 2728520-90-7
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Molecular Weight 434.38
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Formula C19H20F6N4O
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SMILES
OCC1N(C2=NC=NC(N(CC3=CC(C(F)(F)F)=CC(C(F)(F)F)=C3)C)=C2)CCC1
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
Please store the product under the recommended conditions in the Certificate of Analysis.
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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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.
Purity & Documentation
References
Calculators
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
WNK2-IN-1 Related Classifications
HY-189224 Related Classifications
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