TNIK 21 nM (IC50)

Identification of a novel TNIK inhibitor [1]
Having confirmed the safety and feasibility of targeting TNIK, we then screened an in-house kinase-focused compound library and identified a series of quinazoline analogues with high TNIK enzyme-inhibitory activity. Subsequent lead optimization resulted in the identification of NCB-0846 [cis-4-(2-(3H-benzo[d]imidazol-5-ylamino)quinazolin-8-yloxy)cyclohexanol] (Fig. 2a, left). Structure-activity relationship analysis revealed that stereochemistry at the chiral terminal hydroxyl group of the cyclohexane moiety was important for inhibition of the TNIK enzyme. NCB-0846 showed inhibitory activity against TNIK with an half-maximal inhibitory concentration (IC50) value of 21 nM (Fig. 2b), but its diastereomer (named NCB-0970) having the opposite configuration at the terminal hydroxyl group (Fig. 2a, right) showed 13-fold lower TNIK-inhibitory activity (IC50=272 nM) (Fig. 2b). NCB-0846 also inhibited FLT3, JAK3, PDGFRα, TRKA, CDK2/CycA2, and HGK (>80% at 0.1 μM; Supplementary Table 1). NCB-0970 showed a similar inhibitory profile over the human kinome except for STE20 member kinases including TNIK (Fig. 2c). On the basis of these results, we used NCB-0970 as a negative control for TNIK inhibition thereafter.

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NCB-0846 inhibits cancer cell growth in vitro [1]
NCB-0846 showed 6.8-fold higher cell growth-inhibitory activity against HCT116 cells than NCB-0970 under conventional two-dimensional (2D) culture conditions (Fig. 3a). Meanwhile, compared with NCB-0970, NCB-0846 showed much higher (∼20-fold) inhibitory activity against colony formation by the same cells in soft agar (Fig. 3b), indicating that this compound has more potent activity against the clonogenicity of cancer cells.

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TNIK inhibition abrogates colorectal cancer stemness [1]
Active Wnt signalling has been implicated in CSC function. NCB-0846, but not NCB-0970, downregulated the expression of putative colorectal CSC markers: CD44, CD133, and aldehyde dehydrogenase-1 (ALDH1)30,31 (Fig. 5a, left). Flow cytometry analyses revealed that NCB-0846 reduced the proportion of cells showing high expression of CSC surface markers (CD44, CD133, CD166, CD29 and EpCAM) (Supplementary Fig. 7) and ALDH activity (Fig. 5b). CSCs often exhibit the epithelial–mesenchymal transition (EMT) phenotype32. NCB-0846 also reduced the expression of mesenchymal markers (Slug, Snail, Twist, Smad2 and Vimentin; Fig. 5a, right). However, embryonal stem cell markers (Oct4, Nanog and Sox2)33 were not affected (Fig. 5a, right), suggesting that TNIK regulates the stemness of cells committed to intestinal epithelium.

Finally, we explored whether the EMT inhibitory activity of NCB-0846 affects metastasis. A549 cells were treated with TGFβ1 in the absence or presence of either NCB-0846 or NCB-0970 for 48 h in vitro, and then injected into immunodeficient mice (eight per group) via the tail vein. Seven weeks after injection, the mice were sacrificed, and their lung metastases were digitally quantified in tissue sections (Fig. 5a). This animal experiment is used mainly to evaluate the trans-endothelial migration/extravasation capability of cancer cells embolised in peripheral lung vessels immediately after systemic injection. Metastatic lesions occupied 38% of the lung area in mice injected with TGFβ1-treated cells but were significantly (P < 0.001) reduced in the lungs of mice injected with NCB-0846-treated cells (Fig. 5b). We confirmed the complete absence of metastasis by microscopic inspection of tissue sections. In parallel, the average lung weights of mice injected with NCB-0846-treated cells were significantly decreased due to lack of the pulmonary metastatic burden, compared with those of mice injected with cells that had been treated with DMSO (control) or NCB-0970 (Fig. 5c). These results support the notion that inhibition of EMT by NCB-0846 compromises the TGFβ1-induced metastatic potential of lung cancer cells [2].

Mobility shift assay [1]
Enzymatic activity of TNIK was measured by mobility shift assay52 using a QuickScout Screening Assist Kit. The reaction product was quantified using a LabChip EZ Reader II. The IC50 values were calculated from the dose–response curves using nonlinear regression analysis (Fig. 2b).

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 Kinase selectivity profiling [1]
The selectivity of compounds against a panel of 50 human protein kinases was assessed using a non-radiometric assay. Percentage inhibition was determined at an inhibitor concentration of 0.1 M with ATP at the Km concentration (Fig. 2c and Supplementary Table 1).

Evaluation of metastatic potential [2]
Eight-week-old male severe combined immunodeficiency (SCID) mice (C.B-17/Icr-scid) were housed in open-top cages under a 12-h light-dark cycle with free access to standard chow and water ad libitum and maintained in a specific pathogen-free environment. A549 cells were serum-starved for 24 h and stimulated with TGF-β (5 ng/ml) and DMSO (control), NCB-0846 (3 µM) or NCB-0970 (3 µM) for 48 h, and 5.0 × 105 cells in 100 µL of PBS were injected into the anesthetised mice through the tail vein. Seven weeks after the injection of cells treated as indicated, the mice were euthanatised by cervical dislocation, and the digital images of entire lung tissue sections (HE stained) were captured with a BZ-X700 auto-microscope. The areas occupied by metastases were quantified using the Hybrid Cell Count software.

[1]. TNIK inhibition abrogates colorectal cancer stemness. Nat Commun. 2016 Aug 26;7:12586.

[2]. Pharmacological blockage of transforming growth factor-β signalling by a Traf2- and Nck-interacting kinase inhibitor, NCB-0846. Br J Cancer. 2021 Jan;124(1):228-236.

Canonical Wnt/β-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and β-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik(-/-)/Apc(min/+) mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apc(min/+) mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach. [1]

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Background: Metastasis is the primary cause of death in cancer patients, and its management is still a major challenge. Epithelial to mesenchymal transition (EMT) has been implicated in the process of cancer metastasis, and its pharmacological interference holds therapeutic promise. Methods: Traf2- and Nck-interacting kinase (TNIK) functions as a transcriptional coregulator of Wnt target genes. Given the convergence of Wnt and transforming growth factor-β (TGFβ) signalling, we examined the effects of a small-molecule TNIK inhibitor (named NCB-0846) on the TGFβ1-induced EMT of lung cancer cells. Results: NCB-0846 inhibited the TGFβ1-induced EMT of A549 cells. This inhibition was associated with inhibition of Sma- and Mad-Related Protein-2/3 (SMAD2/3) phosphorylation and nuclear translocation. NCB-0846 abolished the lung metastasis of TGFβ1-treated A549 cells injected into the tail veins of immunodeficient mice. The inhibition of EMT was mediated by suppression of the TGFβ receptor type-I (TGFBR1) gene, at least partly through the induction of microRNAs targeting the TGFBR1 transcript [miR-320 (a, b and d) and miR-186].[2]

C21H21N5O2

375.42

2749881-54-5

NCB-0846;1792999-26-8

Typically exists as solids at room temperature

NCB0970; NCB-0970

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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