Axin (disrupts Axin/β-catenin interaction) [1]
β-catenin (stabilizes by inhibiting phosphorylation, no IC50/Ki/EC50 provided) [1]
CK1 (no inhibition, no IC50) [1]
GSK-3β (no inhibition, no IC50) [1]
SKL2001 specifically targets the Wnt/β-catenin signaling pathway by disrupting the Axin/β-catenin interaction. This disruption prevents CK1- and GSK-3β-mediated phosphorylation of β-catenin at residues Ser33/37/Thr41 and Ser45, which would otherwise mark it for proteasomal degradation. SKL2001 does not affect CK1 and GSK-3β enzyme activities, nor does it influence NF-κB or p53 reporter gene activity, demonstrating its selectivity for the Wnt pathway. The compound upregulates the expression of Axin2, a downstream target of the Wnt/β-catenin pathway, confirming pathway activation.

While it has no effect on NF-κB, p53 reporter gene activity, or GSK-3β activity, SKL2001 is an agonist of the Wnt/β-catenin pathway and can also upregulate the expression of Axin2, a downstream target of the Wnt/β-catenin pathway. By stimulating the Wnt/β-catenin pathway, SKL2001 causes osteoblast differentiation (20 and 40 μM) and inhibits preadipocyte differentiation (5, 10 and 30 μM). In 3T3-L1 cells, SKL2001 (5, 10 and 30 μM) stabilizes intracellular β-catenin [1]. HCT116 spheroids are considerably inhibited from proliferating, regardless of cytotoxicity, by SKL2001 (40 μM), and this inhibition is reversible; SKL2001 leads HCT116 spheroids to enter the cell cycle arrest state. E-cadherin expression and round spheroid formation are enhanced by SKL2001 (40 μM) [2].

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SKL2001 (10-30 μM) upregulated TOPflash reporter activity in a dose-dependent manner in HEK293 reporter cells without affecting FOPflash or NF-κB/p53 reporters. [1]
SKL2001 increased cytosolic and nuclear β-catenin protein levels (10 and 30 μM) without changing β-catenin mRNA level. [1]
SKL2001 (10 and 30 μM) inhibited β-catenin phosphorylation at Ser33/37/Thr41 and also at Thr41/Ser45 (unlike LiCl which only inhibited Ser33/37/Thr41). [1]
SKL2001 (up to 10 μM) did not affect GSK-3β-mediated β-catenin phosphorylation in vitro; BIO (5 μM) potently inhibited it. [1]
At 10 μM, SKL2001 did not inhibit any of a panel of recombinant kinases including GSK-3β, while the control inhibitor Novo selectively inhibited GSK-3β. [1]
SKL2001 disrupted the Axin/β-catenin interaction in a GST pull-down assay using purified Axin fragment (362-500) and GST-β-catenin (20 μM SKL2001 competed with Axin for binding to β-catenin). [1]
Co-immunoprecipitation in HEK293 cells showed that SKL2001 (40 μM) prevented the association of Axin with β-catenin. [1]
Overexpression of Axin abrogated SKL2001-mediated CRT activation and β-catenin stabilization. SKL2001 failed to activate CRT in Axin-null SNU475 cells. [1]
In ST2 mesenchymal stem cells, SKL2001 (10-40 μM) dose-dependently increased TOPflash activity, intracellular β-catenin (cytosolic and nuclear), and immunofluorescence staining of β-catenin. [1]
SKL2001 (10-40 μM) increased alkaline phosphatase (ALP) activity in ST2 cells and human primary mesenchymal stem cells. [1]
SKL2001 (10-40 μM) upregulated mRNA expression of osteoblast markers ALP, Runx2, and type I collagen in ST2 cells. [1]
SKL2001 (10-40 μM for 10 days) increased mineralization as assessed by Alizarin red staining in ST2 cells. [1]
In 3T3-L1 preadipocytes, SKL2001 (5-30 μM) inhibited MDI-induced expression of PPARγ and C/EBPα and decreased lipid droplet accumulation (Oil Red O staining). [1]
In vitro studies demonstrate that SKL2001 upregulates β-catenin responsive transcription by increasing intracellular β-catenin protein levels. It promotes osteoblast differentiation at concentrations of 20 and 40 μM and inhibits preadipocyte differentiation at 5, 10 and 30 μM in 3T3-L1 cells. SKL2001 (40 μM) significantly inhibits the proliferation of HCT116 colon cancer spheroids through reversible cell-cycle arrest, without cytotoxicity. It enhances E-cadherin expression and promotes round spheroid formation, indicating its role in maintaining epithelial characteristics. The compound shows no effect on NF-κB or p53 reporter activity.

In vivo, SKL2001 has been shown to reduce cell necrosis and inflammatory cytokines while activating the Wnt/β-catenin pathway in a rat model of caerulein-induced acute pancreatitis. Osteocytes with Wnt signaling activated by SKL2001 provide a safe osteogenic microenvironment for osteoblast differentiation. The compound's ability to activate Wnt signaling in vivo supports its potential therapeutic applications in bone-related disorders and inflammatory conditions. Animal studies have demonstrated that SKL2001 can modulate mesenchymal stem cell differentiation toward the osteogenic lineage while suppressing adipogenesis.

In vitro kinase assay: Purified GST-β-catenin (100 ng) was incubated with purified GSK-3β and CK1 in the presence of indicated concentrations of SKL2001 or BIO (5 μM). Samples were analyzed by western blotting with anti-phospho-β-catenin (Ser33/37/Thr41) antibody; the blot was reprobed with anti-β-catenin antibody as a loading control. [1]
Recombinant kinase activity profiling: Kinase assays were performed by Millipore's KinaseProfiler following the manufacturer's protocols. SKL2001 was assayed at 10 μM against a panel of recombinant kinases; Novo (1-(4-aminofurazan-3-yl)-5-dialkylaminomethyl-1H-[1,2,3]triazole-4-carboxylic acid) was used as a selective GSK-3β inhibitor control. [1]
GST pull-down assay: Purified β-catenin-GST was mixed with 40 μM SKL2001 in ADBII buffer containing pre-equilibrated glutathione-Sepharose 4B beads, incubated at 4°C for 1 h with gentle rotation. Indicated amounts of Axin fragment (362-500) were added and incubated for 2 h at 4°C. Beads were centrifuged, washed five times with PBS, and resuspended in LDS loading buffer. Bound proteins were analyzed by western blotting for β-catenin and silver staining for Axin. [1]
Silver staining: SDS-PAGE gel was placed in fixing solution (50% methanol, 12% acetic acid) for 90 min, washed with 50% ethanol (2×20 min), incubated in sensitization solution (0.02% Na2S2O3) for 1 min, rinsed three times in water, incubated in silver nitrate solution (1% AgNO3 and 0.75 ml/l formalin (37%)) for 20 min, rinsed twice in water, developed in develop solution (Na2CO3, 0.0004% Na2S2O3, 0.5 ml/l formalin (37%)) until bands appeared, and stopped with 1% acetic acid. [1]
In cell-free biochemical assays, SKL2001 disrupts the Axin/β-catenin interaction, which is a critical step for CK1- and GSK-3β-mediated phosphorylation of β-catenin at Ser33/37/Thr41 and Ser45. This disruption prevents β-catenin phosphorylation and subsequent proteasomal degradation while leaving the enzymatic activities of CK1 and GSK-3β unaffected. The assay typically involves purified proteins and measures the binding affinity between Axin and β-catenin in the presence of increasing concentrations of SKL2001 to determine the compound's inhibitory effect on this protein-protein interaction.

HEK293 reporter cell line generation: HEK293 cells stably transfected with hFZ-1 expression plasmid and TOPflash (β-catenin/Tcf-dependent luciferase reporter) were used for screening. Control cells were stably transfected with FOPflash (mutated β-catenin/Tcf-binding elements). Cells were inoculated into 384-well plates at 10,000 cells/well, grown for 24 h, then compounds added at 20 μM final concentration. After 15 h, firefly luciferase activity was assayed. [1]
Luciferase assay: Cells were co-transfected with TOPflash or FOPflash and pCMV-RL plasmids. After treatment with SKL2001 for 15 h, luciferase activity was measured using the Dual Luciferase Assay Kit. [1]
Western blot analysis: Cytosolic and nuclear fractions were prepared. Proteins were separated by 4-12% gradient SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% nonfat milk, probed with anti-β-catenin, anti-phospho-β-catenin (Ser33/37/Thr41), anti-phospho-β-catenin (Thr41/Ser45), anti-Axin, or anti-actin antibodies, then incubated with HRP-conjugated secondary antibodies and visualized using ECL system. [1]
Semi-quantitative RT-PCR: Total RNA was isolated with Trizol reagent. cDNA synthesis, reverse transcription, and PCR were performed. Amplified DNA was separated on 2% agarose gels and stained with ethidium bromide. Primers for β-catenin, GAPDH, ALP, Runx2, type I collagen were used. [1]
Immunofluorescence analysis: ST2 cells cultured on glass chamber slides were treated with DMSO or SKL2001 for 15 h, washed with PBS, fixed with 4% formaldehyde, permeabilized in 0.3% Triton X-100, blocked in 4% bovine serum albumin for 1 h, stained with anti-β-catenin antibody, and analyzed by confocal microscopy. [1]
Alkaline phosphatase (ALP) activity assay: ST2 cells were treated with vehicle or SKL2001 for 72 h. ALP activity was measured in cell lysates using the Alkaline Phosphatase Opt Kit and normalized to protein content. [1]
Alizarin red staining: ST2 cells were treated with SKL2001 at indicated concentrations for 10 days, fixed in 50% ethanol, and stained with 1% Alizarin red. [1]
Oil Red O staining: 3T3-L1 preadipocytes were differentiated with MDI (dexamethasone and insulin) in the presence of SKL2001 for 7 days. Cells were rinsed in PBS, fixed in 3.7% paraformaldehyde for 1 h at 4°C, stained with 1% Oil Red O in 60% isopropanol for 10 min, differentiated with 60% isopropanol, and visualized under phase-contrast microscope. [1]
Immunoprecipitation: HEK293 cells were transiently transfected with β-catenin and Axin-flag, treated with SKL2001 (40 μM) for 15 h. Cell lysates (200 μg) were incubated with 1 μg antibody and protein A-agarose in buffer A (10 mM HEPES pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) overnight at 4°C. Immunocomplexes were washed five times with PBS, boiled in SDS-PAGE loading buffer, and detected by western blotting. [1]
In cellular assays, HEK293 reporter cells containing a β-catenin-responsive firefly luciferase construct are inoculated into 384-well plates at 10,000 cells per well and grown for 24 hours. Test compounds from chemical libraries (approximately 270,000 compounds) are added at a final concentration of 20 μM. After 15 hours of incubation, the plates are assayed for firefly luciferase activity to quantify Wnt pathway activation. SKL2001 treatment results in increased luciferase activity, confirming its agonist activity at the Wnt/β-catenin pathway.

In animal studies, rats are divided into groups including control, caerulein, SKL2001 + caerulein, and SKL2001 + control to evaluate the protective role of SKL2001 in caerulein-induced acute pancreatitis. AR42J cells and rats are treated to assess the compound's anti-inflammatory and tissue-protective effects. Pharmacokinetic parameters are derived from plasma concentration-time curves after oral administration. The compound is typically formulated in 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% saline at 2 mg/mL for in vivo administration.

Pharmacokinetic studies in rats after oral administration of 15 mg/kg/day for seven consecutive days and in beagle dogs after single oral administration of 50 mg/kg show fast absorption. Oral administration in rats at 50 mg/kg/day combined with SRF for seven consecutive days has also been evaluated. The compound's logP is 0.37, indicating moderate hydrophilicity. SKL2001 is soluble in DMSO (242.5 mg/mL) and ethanol (53 mg/mL), but has poor aqueous solubility (<1 mg/mL). The compound should be stored as a powder at -20°C for up to 3 years.

SKL2001 is classified as a toxic compound containing a pharmaceutically active ingredient and should only be handled by personnel trained in handling potent active pharmaceutical ingredients. At 40 μM, SKL2001 significantly inhibits HCT116 spheroid proliferation without cytotoxicity. It may decompose and emit toxic fumes under fire conditions. Standard safety precautions for handling research chemicals apply, including the use of appropriate personal protective equipment and working in a well-ventilated fume hood.

[1]. Small molecule-based disruption of the Axin/β-catenin protein complex regulates mesenchymal stem cell differentiation. ell Res. 2012 Jan;22(1):237-47.

[2]. SKL2001 suppresses colon cancer spheroid growth through regulation of the E-cadherin/β-Catenin complex. Biochem Biophys Res Commun. 2017 Nov 25;493(3):1342-1348.

Wnt-β-catenin pathway agonist; structure described in the first article.

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SKL2001 is a novel small-molecule agonist of the Wnt/β-catenin pathway. Its mechanism of action involves direct disruption of the Axin/β-catenin protein complex, independent of GSK-3β inhibition. Molecular modeling predicted that SKL2001 binds to β-catenin at the same site as Axin via interaction with β-catenin residues Phe253 and Lys292. By preventing Axin from binding to β-catenin, SKL2001 inhibits CK1- and GSK-3β-mediated phosphorylation of β-catenin at Ser45 and Ser33/37/Thr41, leading to β-catenin stabilization and nuclear accumulation, and subsequent activation of downstream target genes (e.g., Axin2). In mesenchymal stem cells, this results in promotion of osteoblastogenesis (increased ALP, Runx2, collagen I, and mineralization) and suppression of adipocyte differentiation (reduced PPARγ, C/EBPα, and lipid accumulation). SKL2001 can be used as a controllable reagent for investigating biological processes involving the Wnt/β-catenin pathway, such as cell differentiation, stem cell renewal, and tissue regeneration, and represents a potential therapeutic candidate for diseases associated with abnormal regulation of this pathway (e.g., bone disorders). An inactive derivative, SKL11324, did not disrupt Axin/β-catenin interaction nor activate the pathway. [1]
SKL2001 is a selective Wnt/β-catenin pathway activator that disrupts the Axin/β-catenin interaction. It suppresses colon cancer spheroid growth via regulation of the E-cadherin/β-catenin complex. The compound has been investigated as a potential safe targeting treatment with fewer side effects for diseases like osteoporosis. Its molecular formula is C14H14N4O3 with a molecular weight of 286.29 g/mol. SKL2001 has been cited in multiple publications in top journals including Nature, Cell, and Science. No clinical trials or approved marketing status have been identified for this research compound; it is intended for research use only.

C14H14N4O3

286.291

286.106

C, 58.74; H, 4.93; N, 19.57; O, 16.77

909089-13-0

16003447

White to off-white solid powder

1.4±0.1 g/cm3

595.7±50.0 °C at 760 mmHg

314.1±30.1 °C

0.0±1.7 mmHg at 25°C

1.655

0.37

1

5

6

21

355

0

O=C(C1C=C(C2=CC=CO2)ON=1)NCCCN1C=CN=C1

PQXINDBPUDNMPE-UHFFFAOYSA-N

InChI=1S/C14H14N4O3/c19-14(16-4-2-6-18-7-5-15-10-18)11-9-13(21-17-11)12-3-1-8-20-12/h1,3,5,7-10H,2,4,6H2,(H,16,19)

5-(Furan-2-yl)-N-(3-imidazol-1-ylpropyl)-1,2-oxazole-3-carboxamide

SKL2001Wnt Agonist II SKL-2001 SKL 2001

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 96.66 mg/mL (~337.63 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.73 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.73 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)