TNKS2: 2 nM (IC50)
TNKS1: 5 nM (IC50)
ARTD2: 479 nM (IC50)
ARTD1: 5500 nM (IC50)

IC50 values for XAV-939 against TNKS1 and TNKS2 are 5 nM and 2 nM, respectively[1]. For three or ten days, XAV-939 (0.3–30 μM) improves the osteoblastic development of hBMSCs [2]. By causing SH3BP2 to accumulate, XAV-939 (3 μM) stimulates the osteoblastic differentiation of hMSCs [2]. During osteoblast development, XAV-939 (3 μM; 10 days) increases the expression of OPG and decreases the expression of RANKL in hBMSC cells [2]. XAV-939 stimulates the production of SFRP3 and SFRP4, while suppressing Wnt/β-catenin signaling [3].

In vivo, XAV-939 restores cartilage degeneration brought on by mechanical stress [3].XAV-939 ameliorated OA severity associated with reduced cartilage degeneration and synovitis in vivo.[4]

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In order to assess whether ablation of Wnt activity can ameliorate the severity of OA in the knee joint, 10-week-old male mice underwent DMM surgery. Three weeks following surgery, the mice were treated with intra-articular injections of either saline control or small-molecule Wnt inhibitor, XAV-939, every 10 days. Knee joints were collected 10 weeks after surgery, 10 days following the last injection (Figure 1A). We assessed the temporal and spatial changes in canonical Wnt/β-catenin signaling by performing immunofluorescence of joint sections using an antibody to β-catenin, a marker of Wnt signaling, and costained with periostin, a stromal/fibroblast marker. Prior to injury, minimal staining was noted; however, after injury, β-catenin was strongly upregulated in the knee joint, particularly in the synovium (Figure 1B), and was attenuated after XAV-939 treatment (Figure 1C). The data demonstrated a striking and dynamic increase in canonical Wnt signaling in the synovium, and the enhanced staining was distinctly colocalized with periostin. Isotype controls showed minimal signal[4].

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In vivo results showed that iOVD-induced mechanical stress in the TMJ disrupted mandible growth, induced OA-like changes in TMJ cartilage, and increased OA-related cytokine expression. In addition, iOVD activated Wnt/β-catenin signaling and suppressed Sfrp1, Sfrp3, and Sfrp4 expression in condylar cartilage. Moreover, in vitro study showed that stress disrupted homeostasis, activated Wnt/β-catenin signaling and inhibited SFRP3 and SFRP4 expression in chondrocytes. Suppression of Wnt/β-catenin signaling with XAV-939 promoted SFRP3 and SFRP4 expression and rescued mechanical stress-induced cartilage degeneration in vivo and in vitro. Conclusions: This work suggests that mechanical stress reduces SFRPs expression both in vivo and in vitro and promotes TMJOA via Wnt/β-catenin signaling. Suppression of Wnt/β-catenin signaling promotes SFRPs expression, especially SFRP3 and SFRP4 expression, and rescues mechanical stress-induced cartilage degeneration. Wnt/β-catenin signaling and SFRPs may represent potential therapeutic targets for TMJOA. Keywords: Mechanical stress; Secreted frizzled-related proteins (SFRPs); Temporomandibular joint osteoarthritis (TMJOA); Wnt/β-catenin.[3]

Screening of Inhibitors and Measurement of Inhibitor Potencies [1]
Flavone derivatives with only one substitution were identified by searching commercially available compound libraries and purchased from different vendors through Molport. The compounds were stored at −20 °C in DMSO and were diluted in the TNKS1 assay buffer before adding them into the reaction mixtures. The compounds were tested at 10 and 1 μM in duplicate. Compound controls were used in this screening to exclude the effect of compound fluorescence and quenching. Inhibition potencies were measured for the inhibitors that had IC50 values below 10 μM based on the two-point initial screening. IC50 values were measured using half log dilutions, and reactions were carried out three individual times in quadruplicate for TNKS1. The incubation time was adjusted so that substrate conversion was more than 50% in the case of screening and less than 30% in the case of IC50 measurement. Dose–response curves were fitted using four parameters with Graphpad Prism (version 5.0 for Windows). [1]

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Profiling of the Inhibitors [1]
The best tankyrase inhibitors identified were also profiled against six other human ARTD family members using the homogeneous activity assay described above. Incubation times and conditions varied for each enzyme based on optimization carried out previously. The substrate NAD+ concentration was 250 or 500 nM in the profiling assays. In order to obtain a robust signal, the incubation time was adjusted so that substrate conversion was more than 50% in each case. In order to efficiently evaluate the selectivity of the compounds, they were profiled at 1 μM. DMSO, compound, and protein controls were used with all the enzymes to exclude or correct for the effects of DMSO, autofluorescence, and quenching of the fluorescence.

Cell Viability Assay[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 0.3, 3, and 30 μM
Incubation Duration: 3 days
Experimental Results: demonstrated no significant effect on proliferation at day 1, 2, and 3 at dose of 0.3 and 3 μM but inhibited hMSCs cell proliferation on day 3 at dose of 30 μM.

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Apoptosis Analysis[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 3 μM
Incubation Duration: 3 days
Experimental Results: demonstrated a minute percentage of cell death (apoptosis and necrosis ) in the XAV-939-treated hBMSC RT-PCR[2]
Cell Types: hMSC-TERT cell line
Tested Concentrations: 3 µM
Incubation Duration: 10 days
Experimental Results: Upregulated gene expression of osteoblast-associated gene markers including: ALP, COL1A1, RUNX2, and OC.

All mice were obtained from The Jackson Laboratory. Ten-week-old male C57BL/6J mice were subjected to DMM surgery to induce OA as described previously. Three weeks after surgery, five intra-articular injections of XAV-939 (0.4 mM) or saline were administered at 10-day intervals, with a total volume of 5 μl. Knee joints from control (n = 8) and Wnt inhibitor mice (n = 8) were collected at 10 weeks after surgery.[4]

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Researchers investigated the progression of mechanical stress-induced TMJOA using an in vivo model via modified increased occlusal vertical dimension (iOVD) malocclusion and an in vitro model in which isolated chondrocytes were subjected to mechanical stress. The effects of inhibition of Wnt/β-catenin signal on TMJOA induced by mechanical stress were studied by in vitro drug added and in vivo intra-articular injection of XAV-939. TMJOA progression, Wnt/β-catenin signaling and SFRPs was assessed by Cone beam computed tomography (CBCT) analysis, histochemical and immunohistochemical (IHC) staining, quantitative real-time PCR (qRT-PCR), Western blotting (WB), and immunofluorescence (IF) staining.[3]

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2.5 mg/kg; i.p. injection

Mouse model

[1]. Discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity. J Med Chem. 2013 Oct 24;56(20):7880-9.

[2]. Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells. Sci Rep. 2020 Oct 7;10(1):16746.

[3]. Mechanical stress reduces secreted frizzled-related protein expression and promotes temporomandibular joint osteoarthritis via Wnt/β-catenin signaling. Bone. 2022 Aug;161:116445.

[4]. Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis. JCI Insight. 2018 Feb 8;3(3):e96308.

XAV939 is a thiopyranopyrimidine in which a 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine skeleton is substituted at C-4 by a hydroxy group and at C-2 by a para-(trifluoromethyl)phenyl group. It has a role as a tankyrase inhibitor. It is a thiopyranopyrimidine and a member of (trifluoromethyl)benzenes.

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Tankyrases are ADP-ribosyltransferases that play key roles in various cellular pathways, including the regulation of cell proliferation, and thus, they are promising drug targets for the treatment of cancer. Flavones have been shown to inhibit tankyrases and we report here the discovery of more potent and selective flavone derivatives. Commercially available flavones with single substitutions were used for structure-activity relationship studies, and cocrystal structures of the 18 hit compounds were analyzed to explain their potency and selectivity. The most potent inhibitors were also tested in a cell-based assay, which demonstrated that they effectively antagonize Wnt signaling. To assess selectivity, they were further tested against a panel of homologous human ADP-ribosyltransferases. The most effective compound, 22 (MN-64), showed 6 nM potency against tankyrase 1, isoenzyme selectivity, and Wnt signaling inhibition. This work forms a basis for rational development of flavones as tankyrase inhibitors and guides the development of other structurally related inhibitors.[1]

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Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.[2]

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Osteoarthritis (OA) is a degenerative joint disease involving both cartilage and synovium. The canonical Wnt/β-catenin pathway, which is activated in OA, is emerging as an important regulator of tissue repair and fibrosis. This study seeks to examine Wnt pathway effects on synovial fibroblasts and articular chondrocytes as well as the therapeutic effects of Wnt inhibition on OA disease severity. Mice underwent destabilization of the medial meniscus surgery and were treated by intra-articular injection with XAV-939, a small-molecule inhibitor of Wnt/β-catenin signaling. Wnt/β-catenin signaling was highly activated in murine synovial fibroblasts as well as in OA-derived human synovial fibroblasts. XAV-939 ameliorated OA severity associated with reduced cartilage degeneration and synovitis in vivo. Wnt inhibition using mechanistically distinct small-molecule inhibitors, XAV-939 and C113, attenuated the proliferation and type I collagen synthesis in synovial fibroblasts in vitro but did not affect human OA-derived chondrocyte proliferation. However, Wnt modulation increased COL2A1 and PRG4 transcripts, which are downregulated in chondrocytes in OA. In conclusion, therapeutic Wnt inhibition reduced disease severity in a model of traumatic OA via promoting anticatabolic effects on chondrocytes and antifibrotic effects on synovial fibroblasts and may be a promising class of drugs for the treatment of OA.[4]

C14H11F3N2OS

312.31

312.054

C, 53.84; H, 3.55; F, 18.25; N, 8.97; O, 5.12; S, 10.27

284028-89-3

135418940

White to off-white solid powder

1.5±0.1 g/cm3

429.3ºC at 760 mmHg

213.4ºC

1.634

2.98

1

6

1

21

505

0

S1C([H])([H])C2C(N([H])C(C3C([H])=C([H])C(C(F)(F)F)=C([H])C=3[H])=NC=2C([H])([H])C1([H])[H])=O

KLGQSVMIPOVQAX-UHFFFAOYSA-N

InChI=1S/C14H11F3N2OS/c15-14(16,17)9-3-1-8(2-4-9)12-18-11-5-6-21-7-10(11)13(20)19-12/h1-4H,5-7H2,(H,18,19,20)

2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-3H-thiopyrano[4,3-d]pyrimidin-4(5H)-one

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)

Solubility in Formulation 1: 1.56 mg/mL (5.00 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 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: 1.56 mg/mL (5.00 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 1.56 mg/mL (5.00 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 15.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Solubility in Formulation 1: ～1.6 mg/mL (5.0 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 16 mg/mL DMSO stock solution and add to 400 μL of PEG300, mix well; Then add 50 μL of Tween 80 to the above solution, mix well; Finally, add 450 μL of saline to the above solution, mix well.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.

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Solubility in Formulation 2: ～1.6 mg/mL (5.0 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 16 mg/mL DMSO stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ～1.6 mg/mL (5.0 mM) 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 take 100 μL of 16 mg/mL DMSO stock solution and add to 900 μL of corn oil, mix well.

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Solubility in Formulation 4: ～5 mg/mL (16.0 mM) in 50% PEG300 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.

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Solubility in Formulation 5: ～30 mg/mL (96 mM) in 30% PEG 400+0.5% Tween 80+5% Propylene glycol (add these co-solvents sequentially from left to right, and one by one).

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Solubility in Formulation 6: 5 mg/mL (16.01 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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