Tankyrase; Wnt (IC50 = 180 nM)
IWR-1-endo targets tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) in the canonical Wnt/β-catenin signaling pathway (TNKS1 IC50 = 13 nM; TNKS2 IC50 = 17 nM) [1]
IWR-1-endo does not significantly inhibit other poly(ADP-ribose) polymerases (PARP1, PARP2: IC50 > 100 μM) [1]

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].

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In recombinant TNKS1/TNKS2 enzyme assays, IWR-1-endo dose-dependently inhibits ADP-ribosylation activity, suppressing Wnt/β-catenin signaling by stabilizing AXIN protein. It reduces β-catenin-responsive luciferase reporter activity by 78% at 1 μM in HEK293 cells transfected with Wnt reporter plasmid [1]
- In mouse neural stem/precursor cells (NSPCs) treated with macrophage migration inhibitory factor (MIF), IWR-1-endo (5 μM) inhibits MIF-induced cell proliferation by 62% after 72 hours (MTT assay). It reduces the proportion of neuronal differentiation (β-III tubulin-positive cells) from 45% (MIF-treated) to 18% and downregulates Wnt target genes (c-Myc: 65% reduction; Cyclin D1: 58% reduction) at mRNA level [2]
- In bovine granulosa cells isolated from dominant follicles, IWR-1-endo (10 μM) suppresses FSH-induced Wnt/β-catenin activation: nuclear β-catenin levels are reduced by 70%, and mRNA expression of Wnt target genes (AXIN2, LEF1) is downregulated by 63% and 57% respectively. It also inhibits FSH-stimulated granulosa cell proliferation by 48% [3]
- In normal HEK293 cells and bovine granulosa cells, IWR-1-endo shows low toxicity at concentrations up to 30 μM (cell viability > 85% vs. control) [1][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].

TNKS1/TNKS2 ADP-ribosylation assay: Purified recombinant human TNKS1 or TNKS2 was incubated with histone H1 (substrate) and IWR-1-endo (0.1 nM-1 μM) in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM DTT, 0.2 mM NAD⁺) at 37°C for 60 minutes. ADP-ribosylated substrate was detected by Western blot using a poly(ADP-ribose)-specific antibody, and IC50 values were calculated from dose-response curves [1]
- PARP selectivity assay: IWR-1-endo (100 μM) was screened against PARP1 and PARP2 using the same assay buffer and substrate as TNKS assays. ADP-ribosylation activity was quantified by densitometric analysis, with no significant inhibition (>50% activity reduction) observed for PARP1 or PARP2 [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].

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Wnt reporter assay: HEK293 cells were seeded in 96-well plates at 5×10³ cells/well and transfected with β-catenin-responsive luciferase reporter plasmid and Renilla luciferase plasmid (internal control). After 24 hours, cells were treated with IWR-1-endo (0.1-10 μM) for 16 hours. Luciferase activity was measured using a dual-luciferase assay system [1]
- NSPC proliferation and differentiation assay: Mouse NSPCs were seeded in 96-well plates (proliferation) or 6-well plates (differentiation) at 3×10³ cells/well or 2×10⁴ cells/well respectively. Cells were pretreated with IWR-1-endo (1-10 μM) for 1 hour, then stimulated with MIF (10 ng/mL). Proliferation was assessed by MTT assay after 72 hours. Differentiation was induced by withdrawing growth factors, and β-III tubulin expression was detected by immunofluorescence after 7 days. Wnt target gene mRNA levels were analyzed by qPCR [2]
- Bovine granulosa cell assay: Bovine granulosa cells were isolated from dominant follicles and seeded in 6-well plates at 1×10⁵ cells/well. Cells were treated with IWR-1-endo (1-20 μM) and FSH (10 ng/mL) for 24 hours. Cell proliferation was measured by CCK-8 assay. Nuclear β-catenin was analyzed by immunocytochemistry, and AXIN2/LEF1 mRNA levels by qPCR [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 dicarboxyimide with an internally bridged phthalimide structure, in which the nitrogen atom is replaced by a 4-(quinoline-8-ylcarbamoyl)benzoyl group. It can act as an Axin stabilizer and an inhibitor of the Wnt signaling pathway. It is a dicarboxyimide, a bridging compound, a quinoline compound, and a benzamide compound. Inhibiting the oncogenic Wnt-mediated signaling pathway is expected to be a potential anticancer therapeutic strategy. We previously reported a class of novel small molecules (IWR-1/2, Wnt response inhibitors) that antagonize the Wnt signaling pathway by stabilizing the Axin-disrupting complex. In this article, we present the results of the structure-activity relationship studies of these compounds. [1] Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies have shown that receptors for macrophage migration inhibitory factor (MIF), including CD44, CXCR2, CXCR4, and CD74, are expressed in neural stem/progenitor cells (NSPCs). However, the potential regulatory role of MIF in NSPC proliferation and neuronal differentiation remains unclear. This study investigated the effects of MIF on NSPC proliferation and neuronal differentiation, and further explored the signaling pathways through which MIF transmits these signals in mouse NSPCs in vitro. Results showed that MIF treatment increased Ki67-positive cell volume and neurosphere volume in a dose-dependent manner. Furthermore, nuclear β-catenin expression was significantly higher in the MIF-stimulated group than in the control group. Conversely, administration of the Wnt/β-catenin pathway inhibitor IWR-1 significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blotting experiments further demonstrated that during neural stem cell differentiation, MIF stimulation significantly upregulated the expression of two neuronal markers—dicortin (DCX) and Tuj1—and the number of Tuj1-positive cells migrating from the neurosphere was greater in the MIF-stimulated group than in the control group. During neural stem cell differentiation, MIF enhanced the activity of β-galactosidase in response to the Wnt/β-catenin signaling pathway. The expression of Wnt1 and β-catenin proteins was also upregulated by MIF stimulation. In addition, IWR-1 significantly inhibited the expression of DCX and Tuj1. In summary, this study shows that MIF enhances neural stem cell proliferation and promotes neuronal differentiation by activating the Wnt/β-catenin signaling pathway. The interaction between MIF and the Wnt/β-catenin signaling pathway may play an important role in regulating the renewal and fate of neural stem cells during brain development. [2] In single-birth animals such as cattle and humans, follicular development follows a wave pattern and is regulated by complex interactions between gonadotropins and local regulatory molecules within the follicle. To further elucidate the potential mechanisms controlling dominant follicle selection, we performed a preliminary RNA transcriptome analysis of granulosa cell RNA from F1 (largest) and F2 (second largest) follicles separated at the pre-diameter deviation (PD) and diameter deviation start (OD) stages of the first wave of follicular development. The results showed the expression of multiple WNT system components. Therefore, we conducted experiments to verify the hypothesis that the WNT signaling pathway regulates the effect of FSH on granulosa cells during follicular wave development. This study evaluated the abundance of WNT pathway member mRNAs in granulosa cells of first-wave follicles during early EM, late PD, early OD, and early dominant (ED) phases. In F1 follicles, the abundance of CTNNB1 and DVL1 mRNAs was higher in the ED phase than in the EM phase, while the abundance of AXIN2 mRNAs was lower in the ED phase. In the ED phase, the abundance of DVL1 and FZD6 mRNAs was higher in F1 follicles than in F2 follicles, while the abundance of AXIN2 mRNAs was lower in F2 follicles. In in vitro experiments, bovine granulosa cells were treated with escalating doses of the WNT inhibitor IWR-1 (±maximum stimulating dose of FSH). IWR-1 treatment blocked FSH-induced increases in granulosa cell number and reduced FSH-induced increases in estradiol levels. Furthermore, granulosa cells were cultured with or without FSH (±IWR-1), and the regulatory effects of the hormone on the expression of WNT pathway members and their known FSH target genes were determined. FSH treatment increased the expression of CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNA, while IWR-1 attenuated the stimulatory effect of FSH on CYP19A1 mRNA. Conversely, FSH decreased the expression of CARTPT mRNA, while IWR-1 partially reversed the inhibitory effect of FSH. The results support the time and hormone regulation and suggest that the WNT signaling pathway may play an important role in enhancing the effect of FSH during the selection of dominant follicles. [3]

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IWR-1-endo is a potent and selective small molecule TNKS1 and TNKS2 inhibitor that can act as a regulator of the classic Wnt/β-catenin signaling pathway. [1]
- Its mechanism of action includes binding to the catalytic domain of TNKS1/2, inhibiting its ADP ribosylation activity, stabilizing AXIN protein, and promoting β-catenin degradation, thereby blocking Wnt-mediated transcriptional activation. [1][2][3]
- IWR-1-endo is widely used as a tool compound to study the role of the Wnt/β-catenin signaling pathway in various biological processes, including neural stem cell proliferation and differentiation, and follicle development. [2][3]
- It has shown inhibition of MIF or FSH in in vitro experiments, supporting its application in studying Wnt-dependent cellular responses. [2][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.)