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Purity: ≥98%
NVP-TNKS656 (also called TNKS-656; NVP-TNKS-656; TNKS 656; NVP-TNKS 656) is a selective, and orally bioactive tankyrase inhibitor with potential antitumor activity. It blocks tankyrase/TNKS2 with an IC50 of 6 nM, and shows > 300-fold selectivity against PARP1 and PARP2.
| Targets |
TNKS2 ( IC50 = 6 nM ); PARP2 ( IC50 = 32 μM )
NVP-TNKS656 targets tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) with over 5,000-fold selectivity versus PARP1 and PARP2. [1] In colorectal cancer cells, NVP-TNKS656 inhibits Wnt/β-catenin signaling by stabilizing AXIN1 and reducing nuclear β-catenin. [2] |
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| Enzyme Assay |
Nicotinamide is detected by quantitative liquid chromatography/mass spectrometry (LC-MS), which is used to track PARP catalytic activity. 384-well Greiner flat-bottom plates are used for the autoPARsylation reactions, which are carried out at room temperature. The final reaction mixture contains 2.5% DMSO and inhibitors with concentrations ranging from 0.0001 to 18.75 μM. The enzymes PARP1, PARP2, GST-TNKS2P, and GST-TNKS1P are utilized at final concentrations of 5, 5, 5, and 2 nM, respectively. Quantitative liquid chromatography/mass spectrometry (LC-MS), which monitors PARP catalytic activity, is used to identify nicotinamide. 384-well Greiner flat-bottom plates are used for the autoPARsylation reactions, which are carried out at room temperature. The final reaction mixture contains 2.5% DMSO and inhibitors with concentrations ranging from 0.0001 to 18.75 μM. Final concentrations of 5, 5, 5, and 2 nM are used for the enzymes PARP1, PARP2, GST-TNKS2P, and GST-TNKS1P, respectively.
Tankyrase autoPARsylation assay: PARP catalytic activity was monitored by quantitative LC-MS detection of nicotinamide. Reactions were performed at room temperature in 384-well plates. The final reaction mixture contained 2.5% DMSO and inhibitors at concentrations ranging from 0.0001 to 18.75 μM. GST-TNKS2P, GST-TNKS1P, PARP1, and PARP2 enzymes were used at final concentrations of 5 nM, 5 nM, 5 nM, and 2 nM respectively. The nicotinamide concentration in supernatants was measured by LC-MS. Percent inhibition was calculated as (control - sample)/(control - background) × 100. [1] Isothermal calorimetry (ITC): Thermodynamic properties of ligand binding were determined at 25°C. Data were collected using a sample buffer of 25 mM HEPES pH 7.4, 150 mM NaCl, with matched DMSO concentration. For NVP-TNKS656, a direct titration of 200 μM compound into 20 μM TNKS1 was performed over 16 injections of 2.5 μL. Individual heat values were plotted against molar ratio, and values for number of binding sites (n), enthalpy change (ΔH), and dissociation constant (KD = 1/KA) were obtained by nonlinear regression. [1] |
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| Cell Assay |
SuperTopFlash (STF) reporter gene assay: Compound activity in inhibiting Wnt ligand signaling was measured using a Wnt-responsive STF luciferase reporter gene assay in HEK293 cells. Percent inhibition was calculated as (maximum Wnt-induced signaling - sample)/(maximum Wnt-induced signaling - background) × 100. Maximum Wnt-induced signaling was the STF signal level induced by 20% Wnt3A CM without compound, and background was the STF signal level without Wnt3A CM or compound. A counter-screen was performed in HEK293T cells expressing a cAMP-response element (CRE) luciferase reporter gene with 10 μM forskolin. [1]
Axin2 protein ELISA: Compound activity in stabilizing Axin2 protein was measured by sandwich ELISA in SW480 cells. Cell lysates were prepared from cells treated with compounds in six-point dilution starting at 10 μM for 24 hours. Anti-Axin2 capture antibody was diluted to 1 μg/mL in carbonate coating buffer pH 9.2, and 100 μL per well was used to coat 96-well plates overnight at 4°C. After washing and blocking with 1% BSA/PBS, 100 μL of cell lysate was added per well and incubated for 2 hours at room temperature. Biotinylated anti-Axin2 antibody was then added, and signal was detected by chemiluminescence using streptavidin-HRP. [1] Apoptosis assay in patient-derived sphere cultures: Cells were seeded as sphere cultures on low-attachment plates. They were pretreated with NVP-TNKS656 (100 nM) or DMSO for 48 hours, then with API2 (20 μM) and/or NVP-BKM120 (2.4 μM) for another 48 hours. Apoptotic cells were determined using Annexin V-eGFP kit and DAPI staining, analyzed by flow cytometry. For immunofluorescence detection of apoptosis in spheres, cells mixed 1:1 with Matrigel were fixed in 4% paraformaldehyde, permeabilized with PBS/1% Triton X-100, blocked, and incubated with primary antibodies against cleaved-CASPASE3, then secondary antibodies and Hoechst 33342. [2] TCF/LEF reporter assay: DLD1 cells stably transfected with a 7TGP vector expressing eGFP under seven TCF/LEF binding sites (7xTOP) were treated with NVP-TNKS656 100 nM for seven days, and eGFP accumulation was measured by flow cytometry. [2] |
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| Animal Protocol |
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| ADME/Pharmacokinetics |
Mouse pharmacokinetic properties of NVP-TNKS656 after intravenous administration (1 mg/kg): AUC 3.46 μM·h, CL 9.7 mL/min/kg, t1/2 1.3 h, Vdss 0.6 L/kg. After oral administration (30 mg/kg): AUC 33.4 μM·h, Cmax 16.8 μM, Tmax 0.4 h, F 32%. After oral administration (100 mg/kg): AUC 183.4 μM·h, Cmax 79.7 μM, Tmax 0.8 h, F 53%. Slight overproportional increase in oral exposure was observed between 30 and 100 mg/kg (dose-normalized AUC for 100 mg/kg was 2-fold higher than for 30 mg/kg). [1]
Free fraction in mouse plasma: 16% as determined by rapid equilibrium dialysis. [1] |
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| Toxicity/Toxicokinetics |
NVP-TNKS656 treatment caused a systemic reduction of nuclear β-catenin content and function in skin and intestine (tissues where Wnt pathway controls homeostasis) but showed no major negative side effects in mice. [2]
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| References | ||
| Additional Infomation |
Mechanism of action: NVP-TNKS656 inhibits tankyrase 1 and 2, leading to stabilization of AXIN1/2 proteins, enhanced activity of the β-catenin destruction complex, reduced free and nuclear β-catenin, and consequent inhibition of Wnt/β-catenin target gene expression. It exhibits an enthalpy-driven binding signature (high enthalpy change) which contributes to its high selectivity against PARP1 and PARP2. [1]
In colorectal cancer, NVP-TNKS656 overcomes resistance to PI3K and AKT inhibitors that is mediated by high nuclear β-catenin. High nuclear FOXO3A content predicts sensitivity to NVP-TNKS656 treatment. Combined treatment with PI3K/AKT inhibitors and NVP-TNKS656 promotes apoptosis in resistant cells. The compound represses FOXO3A/β-catenin target genes (e.g., SLC2A3) and TCF/β-catenin target genes. Nuclear β-catenin and FOXO3A are proposed as predictive biomarkers for response to NVP-TNKS656 and PI3K/AKT inhibitors. [2] |
| Molecular Formula |
C27H34N4O5
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| Molecular Weight |
494.58
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| Exact Mass |
494.253
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| Elemental Analysis |
C, 65.57; H, 6.93; N, 11.33; O, 16.17
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| CAS # |
1419949-20-4
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| Related CAS # |
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| PubChem CID |
136237316
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| Appearance |
White to light yellow solid powder
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| LogP |
2.122
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
36
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| Complexity |
911
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C([H])([H])N1C([H])([H])C([H])([H])C([H])(C(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[H])=O)C([H])([H])C1([H])[H])N(C([H])([H])C1=NC2C([H])([H])C([H])([H])OC([H])([H])C=2C(N1[H])=O)C([H])([H])C1([H])C([H])([H])C1([H])[H]
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| InChi Key |
DYGBNAYFDZEYBA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H34N4O5/c1-35-21-6-4-19(5-7-21)26(33)20-8-11-30(12-9-20)16-25(32)31(14-18-2-3-18)15-24-28-23-10-13-36-17-22(23)27(34)29-24/h4-7,18,20H,2-3,8-17H2,1H3,(H,28,29,34)
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| Chemical Name |
N-(cyclopropylmethyl)-2-[4-(4-methoxybenzoyl)piperidin-1-yl]-N-[(4-oxo-3,5,7,8-tetrahydropyrano[4,3-d]pyrimidin-2-yl)methyl]acetamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.05 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.05 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (10.11mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0219 mL | 10.1096 mL | 20.2192 mL | |
| 5 mM | 0.4044 mL | 2.0219 mL | 4.0438 mL | |
| 10 mM | 0.2022 mL | 1.0110 mL | 2.0219 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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