Tankyrase; Wnt (IC50 = 180 nM)

IWR-1 and XAV939 have comparable pharmacological actions in vitro and function as reversible inhibitors of the Wnt pathway. IWR-1 interacts with Axin to provide its effect, whereas XAV939 directly binds to TNKS[1]. IWR-1 (10 μM) causes the β-catenin disruption complex to stabilize. When IWR-1 (10 μM) is given to the medium together with MIF, the size of the cell colonies is drastically reduced, indicating that IWR-1 inhibits MIF's stimulating effect on NSPC proliferation in all MIF concentration groups. The proliferation of NSPC is strongly inhibited, dose-dependently, by 2, 5, and 10 μM of IWR-1. MIF's stimulating effect on NSPC development into the neuron lineage is inhibited by IWR-1[2]. The stimulatory action of FSH is dose-dependently inhibited by IWR-1 administration in the presence of the maximal stimulatory dose of FSH (0.5 ng/mL), with > 75% inhibition seen at the maximal inhibitory dose of IWR-1 (1 µM). The FSH-induced suppression of granulosa cell CARTPT mRNA expression is partially reversed by IWR-1 treatment[3].

Researchers previously reported that exo-IWR 51 was only active at high concentration.5 Quantitatively, 51 was 25-fold less active than endo-IWR 1 (Figure 2). Interestingly, saturation of the olefin did not affect the activity. Sat-IWR 52 and 1 were equally potent in the in vitro assays (Figure 2). These results indicated that the norbornyl region of 1 could only tolerate subtle steric perturbation. Researchers have also tested the in vivo activity of IWR’s and found that 1 effectively inhibited zebrafish tail fin regeneration. We show herein that the minimum inhibitory concentration of 1 is 0.5 μM (Figure 3). They further demonstrated that the in vivo activity of IWR’s correlated with their in vitro activity. For example, only partial inhibition of fin regeneration was observed with moderate inhibitors 13 and 43. The weak inhibitor 17 only retarded the growth of the tail fin (picture not shown)[1].

For NSPC proliferation experiment, single, dissociated cells were seeded into a 96-well plate at a density of 1×105/ml with different MIF concentration (0, 1, 2, 4, 8, 16, 32ng/ml) with or without IWR-1 (10μM; Sigma, St. Louis, MO). Four days later, observed the neurospheres and took photomicrographs with an invert microscope. Six pups were used in single culture and experiments were repeated 3 times. Analyzed the images by counting the number and measuring the diameter of neurospheres with Image-Pro Plus 5.0 software. Part of the cells were seeded on poly-D-lysine hydrochloride (PLL, molecular weight of 70,000∼150,000) - coated 10-mm glass coverslips in 24-well plates in NSPC medium and immunostained with Ki67 and Hoechst antibodies four days later. For Ki67-immunostaining cells, MIF concentration is 16ng/ml in MIF-stimulated group.[2]

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For NSPC differentiation studies, neurospheres were seeded on poly-D-lysine hydrochloride (PLL, molecular weight of 70,000∼150,000) - coated 10-mm glass coverslips in 24-well plates or flasks with neural differentiation medium containing DMEM/F-12 supplemented with 2% B27 and 2% fetal bovine serum (FBS; Invitrogen), with or without MIF (16ng/ml) or IWR-1 (1 or 10μM). Seven to ten days later, stopped differentiation and fixed the cells with 4% paraformaldehyde for staining or collected the cells in RIPA buffer for Western blot[2].

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The first experiment examined the effect of the WNT signaling inhibitor IWR-1 on basal and FSH-induced estradiol production and cell numbers. The WNT inhibitor stabilizes the interaction of AXIN2 with CTNNB1 leading to degradation of CTNNB1 and inhibition of the canonical WNT pathway. Treatments consisted of culture medium with DMSO (diluent control group) or medium containing 0.1, 1.0 or 10 µM of IWR-1 with or without the addition of maximal stimulatory dose of FSH (0.5 ng/ml; NHPP) for 6 days with 12 wells per treatment in each replicate experiment. Media was changed every 2 days. On the 6th day of culture, media was removed and stored at −20°C until analysis for estradiol concentrations and the cells were washed, trypsinized and counted using a Coulter counter (Beckman Coulter) set to count cells between 5 and 20 µm in size, as previously described [15]. The experiment was replicated 4 times using ovaries obtained on different days.[3]
In the second experiment, the effect of FSH and maximal inhibitory dose of IWR-1 on mRNA abundance for select WNT pathway members and other modulators of FSH action were determined. In this experiment, granulosa cells were treated (24 wells per treatment) with DMSO (vehicle control) or the maximally effective dose of IWR-1 (1 µM) in the presence or absence of FSH (0.5 ng/ml). On the 6th day of culture, media was removed and stored at −20°C until analysis for estradiol concentrations and the cells were lysed and preserved at −80°C until processed for total RNA isolation. The experiment was replicated 4 times using ovaries obtained on different days.[3]
In the third experiment, the effect of IWR-1 treatment on CTNB1 and AXIN2 protein abundance was determined. For this experiment, granulosa cells were isolated and cultured as described for experiment 2. On the 6th day of culture, media was removed and stored at −20°C until analysis for estradiol concentrations. The cells were then washed, aspirated from the wells and centrifuged at 3000 g for 5 min. The cell pellet was then snap frozen in liquid nitrogen and preserved at −80°C until Western blot analysis[3].

[1]. Structure-activity relationship studies of small-molecule inhibitors of Wnt response. Bioorg Med Chem Lett. 2009 Jul 15;19(14):3825-7.

[2]. Macrophage migration inhibitory factor promotes proliferation and neuronal differentiation of neural stem/precursor cells through Wnt/β-catenin signal pathway. Int J Biol Sci. 2013 Nov 28;9(10):1108-20.

[3]. Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection. PLoS One. 2014 Jun 17;9(6):e100201.

IWR-1-endo is a dicarboximide having an endo bridged phthalimide structure, substituted at nitrogen by a 4-(quinolin-8-ylcarbamoyl)benzoyl group. It has a role as an axin stabilizer and a Wnt signalling inhibitor. It is a dicarboximide, a bridged compound, a member of quinolines and a member of benzamides.

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Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, inhibitors of Wnt response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure-activity relationship studies of these compounds.[1]

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Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.[2]

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Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.[3]

C25H19N3O3

409.44

409.142

, 73.34; H, 4.68; N, 10.26; O, 11.72

1127442-82-3

44483163

Off-white to yellow solid powder

1.4±0.1 g/cm3

643.9±55.0 °C at 760 mmHg

343.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.741

2.65

1

4

3

31

772

4

ZGSXEXBYLJIOGF-UHFFFAOYSA-N

InChI=1S/C25H19N3O3/c29-23(27-19-5-1-3-14-4-2-12-26-22(14)19)15-8-10-18(11-9-15)28-24(30)20-16-6-7-17(13-16)21(20)25(28)31/h1-12,16-17,20-21H,13H2,(H,27,29)

4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide

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: ≥ 2.5 mg/mL (6.11 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 (6.11 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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: ≥ 2.5 mg/mL (6.11 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.)