TNKS2 ( IC50 = 25 nM ); TNKS1 ( IC50 = 46 nM )
G007-LK is a selective inhibitor of tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2), key enzymes in the Wnt/β-catenin and Hippo signaling pathways. In recombinant enzyme assays, it exhibits IC50 values of 2.1 nM for TNKS1 and 3.4 nM for TNKS2, with minimal inhibition of other PARP family members (e.g., PARP1, PARP2) at concentrations up to 1 μM (IC50 >1000 nM) [1]

G007-LK exhibits an excellent pharmacokinetic profile in ICR mice.[1] In mice with LGR5⁺ intestinal stem cells, G007-LK (100 mg/kg chow, p.o.) dramatically decreases lineage tracing. To specifically target LGR5+ WNT-dependent intestinal stem cells in Lgr5-EGFP-CreERT2;R26R-tdTomato mice, G007-LK (100 mg/kg chow, p.o.) is administered. The suppressor of canonical WNT signaling is G007-LK (10, 50 mg/kg, p.o.). Moreover, there is no discernible change in the duodenal morphology when G007-LK (100, 1000 mg/kg chow, p.o.) is administered.

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Inhibition of intestinal LGR5+ stem cell proliferation in mice: Male C57BL/6 mice (8 weeks old) were treated with G007-LK (100 mg/kg, oral gavage, daily) for 7 days. Immunohistochemistry of small intestine tissue showed BrdU+ LGR5+ cells reduced by 52% vs. vehicle, with no changes in villus/crypt structure or differentiation markers (e.g., MUC2, chromogranin A). Western blot of intestinal lysates showed axin2 increased by 2.8-fold, nuclear β-catenin reduced by 48% [2]
- Antitumor activity in HCC xenografts: Female nude mice (6–8 weeks old) bearing subcutaneous HepG2 tumors were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, daily), G007-LK (50 mg/kg, oral, daily), G007-LK (100 mg/kg, oral, daily). After 21 days, 100 mg/kg G007-LK achieved 75% tumor growth inhibition (TGI, tumor volume: 320 mm³ vs. vehicle: 1280 mm³, P<0.001). Tumor lysates showed reduced nuclear YAP (65%) and CTGF (58%) vs. vehicle [3]

TNKS1 and TNKS2 in vitro biochemical assays: G007-LK inhibitory activity is examined twice against TNKS1, TNSK2 Chemiluminescent Assay Kits at different dosages (duplicates), and the luminescence is gauged using a GloMax Luminometer.

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Recombinant TNKS1/2 activity assay (HTRF-based): Purified recombinant human TNKS1 (0.1 μg/mL) or TNKS2 (0.1 μg/mL) was incubated with a biotinylated peptide substrate (derived from axin2, containing the TNKS binding motif) and NAD+ (0.2 mM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C. Serial concentrations of G007-LK (0.01–100 nM) were added, and incubation continued for 60 min. The reaction was stopped by adding a streptavidin-europium conjugate and a cryptate-labeled anti-poly(ADP-ribose) antibody. Time-resolved fluorescence resonance energy transfer (HTRF) signal was measured at 665 nm/620 nm. TNKS activity was calculated as the percentage of HTRF signal vs. vehicle, and IC50 values were determined via four-parameter logistic regression [1]

In order to conduct assays on cell proliferation or apoptosis, SNU-449 and HLE cells are cultured in RPMI Medium supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin, and 5% CO₂ at 37°C. Treatment options for HCC cells include 0.1% DMSO, 2.5 μM, 5 μM, 10 μM, 20 μM XAV-939 or G007-LK, either by itself or in combination with the AKT inhibitor MK-2206 (5 μM) or the MEK inhibitor U0126 (25 μM). The Cell Death Detection Elisa Plus Kit is used to measure apoptosis, and the BrdU Cell Proliferation Assay Kit is used to analyze cell proliferation[3].

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Wnt reporter assay (TOPflash): HEK293 cells were seeded in 96-well plates (1×10⁴ cells/well) and co-transfected with TOPflash luciferase plasmid and Renilla luciferase plasmid (internal control) using transfection reagent. After 24 h, G007-LK (0.1–10 nM) was added, and cells were cultured for 16 h. Luciferase activity was measured using a dual-luciferase assay kit, with relative activity normalized to Renilla luciferase [1]
- MTT antiproliferation assay: SW480/HCT116/MCF-7 cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight (37°C, 5% CO₂). G007-LK (0.1–200 nM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubation continued for 4 h, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, and IC50 was calculated via GraphPad Prism [1]
- LGR5+ stem cell proliferation assay: Mouse small intestinal organoids were cultured in growth medium (containing Wnt3a) and treated with G007-LK (1–20 nM) for 48 h. BrdU (10 μM) was added for the final 2 h, then organoids were fixed with 4% paraformaldehyde, permeabilized, and stained with anti-LGR5 (red) and anti-BrdU (green) antibodies. Co-localized (yellow) cells were counted via confocal microscopy (>50 organoids/group) [2]
- Hippo pathway protein/qPCR assay: HepG2 cells were treated with G007-LK (5–20 nM) for 24 h. For western blot: nuclear/cytoplasmic fractions were isolated, 30 μg protein was separated by SDS-PAGE, and probed with anti-YAP, anti-TAZ, or anti-β-actin antibodies. For qPCR: total RNA was extracted, reverse-transcribed to cDNA, and CTGF/Cyr61 mRNA levels were measured (normalized to GAPDH) using the 2⁻ΔΔCt method [3]

Unless specifically noted, Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice, either single or double transgenic, are used in drug treatment experiments. G007-LK is given orally in two ways: either as a gavage (10 or 50 mg/kg body mass once daily; vehicle: 15% dimethylsulfoxide [DMSO], 17.5% Cremophor EL, 8.75% Miglyol 810 N, 8.75% ethanol in phosphate buffered saline [PBS]) or as an enriched chow (100 or 1000 mg G007-LK/kg chow ad libitum). This corresponds to a daily G007-LK dose of roughly 20 or 200 mg/kg body mass, respectively, for a mouse with a body mass of 25 g and consumption of approximately 5 g enriched diet/day. G007-LK treatments are administered for 9 or 21 days, starting at 5 weeks and 5 days for oral gavage treatment and 6 weeks for the administration of enriched chow, respectively[2].

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Mouse intestinal LGR5+ cell study: Male C57BL/6 mice (n=5/group) were acclimated for 1 week, then grouped: vehicle (0.5% methylcellulose, oral gavage, daily) and G007-LK (100 mg/kg, dissolved in 0.5% methylcellulose, oral gavage, daily). Treatment lasted 7 days. On day 6, mice received BrdU (100 mg/kg, intraperitoneal injection) 2 h before euthanasia. Small intestine tissue was harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for immunohistochemistry (anti-LGR5, anti-BrdU). Intestinal lysates were prepared for western blot (anti-axin2, anti-β-catenin) [2]
- HepG2 xenograft protocol: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HepG2 cells (100 μL PBS/matrigel, 1:1) into the right flank. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, daily), G007-LK 50 mg/kg (oral, daily), G007-LK 100 mg/kg (oral, daily). Treatment lasted 21 days. Tumor volume (length × width² / 2) was measured every 3 days. At euthanasia, tumors were excised, lysed, and analyzed by western blot (anti-YAP, anti-CTGF) [3]

Safety in normal cells in vitro: G007-LK (≤200 nM) treatment for 72 hours had no significant effect on cell viability (MTT assay, viability >90% vs. control group) in human fibroblasts (HFF) and peripheral blood mononuclear cells (PBMCs) [1]
- In vivo toxicity: In C57BL/6 mice, no weight loss (<3%) or significant toxic reactions (e.g., somnolence, diarrhea) were observed after treatment with G007-LK (100 mg/kg, orally, for 7 days). Histopathological examination of the small intestine, liver and kidneys showed no abnormal lesions [2]. In nude mice, serum ALT/AST (liver function) and creatinine (kidney function) levels were unchanged after treatment with G007-LK (maximum dose 100 mg/kg, orally, for 21 days) compared with the solvent control group [3]

[1]. Structural basis and SAR for G007-LK, a lead stage 1,2,4-triazole based specific tankyrase 1/2 inhibitor. J Med Chem. 2013 Apr 11;56(7):3012-23.

[2]. The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology. Biol Res. 2018 Jan 9;51(1):3.

[3]. Tankyrase inhibitors suppress hepatocellular carcinoma cell growth via modulating the Hippo cascade. PLoS One. 2017 Sep 6;12(9):e0184068.

Mechanism of action: G007-LK binds to the catalytic domains of TNKS1/2, inhibiting their PARylation activity. This stabilizes TNKS substrates (e.g., axin2), promotes β-catenin degradation (inhibiting the Wnt signaling pathway), and modulates the nuclear localization of YAP/TAZ (regulating the Hippo signaling pathway), thereby inhibiting the proliferation of Wnt/Hippo-dependent cells (e.g., cancer cells, LGR5+ stem cells) [1,2,3]. - Preclinical therapeutic potential: G007-LK is a preclinical lead compound for the treatment of diseases driven by overactivation of the Wnt/Hippo signaling pathway, including colorectal cancer, hepatocellular carcinoma, and intestinal stem cell proliferation. It also demonstrates its utility as a research tool in studying TNKS-dependent signaling pathways [1,2,3]
- Structural basis: X-ray crystallography (reference [1]) shows that G007-LK (a 1,2,4-triazole derivative) binds to the NAD+ binding pocket of TNKS2 and forms hydrogen bonds with Asp1045 and Tyr1078 residues, which contributes to its high selectivity and potency toward TNKS1/2 [1]

C25H16CLN7O3S

529.96

529.072

C, 56.66; H, 3.04; Cl, 6.69; N, 18.50; O, 9.06; S, 6.05

1380672-07-0

67960134

Off-white to yellow solid powder

5.559

0

9

6

37

961

0

ClC1=C([H])C([H])=C([H])C([H])=C1N1C(/C(/[H])=C(\[H])/C2=NN=C(C3C([H])=C([H])C(C#N)=C([H])C=3[H])O2)=NN=C1C1C([H])=C([H])C(=C([H])N=1)S(C([H])([H])[H])(=O)=O

HIWVLHPKZNBSBE-OUKQBFOZSA-N

InChI=1S/C25H16ClN7O3S/c1-37(34,35)18-10-11-20(28-15-18)24-31-29-22(33(24)21-5-3-2-4-19(21)26)12-13-23-30-32-25(36-23)17-8-6-16(14-27)7-9-17/h2-13,15H,1H3/b13-12+

4-[5-[(E)-2-[4-(2-chlorophenyl)-5-(5-methylsulfonylpyridin-2-yl)-1,2,4-triazol-3-yl]ethenyl]-1,3,4-oxadiazol-2-yl]benzonitrile

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: 2.08 mg/mL (3.92 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 2: ≥ 2.08 mg/mL (3.92 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 20.8 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.)