NCB-0846

Biological Activity

In vitro: NCB-0846 blocks Wnt signalling and shows marked anti-tumour and anti-CSC activities. NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 shows inhibitory activity against TNIK with an half-maximal inhibitory concentration (IC50) value of 21 nM. It also inhibits FLT3, JAK3, PDGFRα, TRKA, CDK2/CycA2, and HGK (>80% at 0.1 μM). NCB-0846 induces faster migration of TCF4 phosphorylated by TNIK within a concentration range of 0.1-0.3 μM and completely inhibits the phosphorylation of TCF4 at a concentration of 3 μM. Furthermore, NCB-0846 blocks the auto-phosphorylation of TNIK. NCB-0846 inhibits the TCF/LEF transcriptional activity of Wnt3a-treated HEK293 and HCT116 (carrying CTNNB1 mutation) and DLD-1 (carrying APC mutation) colorectal cancer cells. NCB-0846 reduces the expression of the Wnt target genes AXIN2 and MYC as well as that of TNIK, but the expression of CCND1 is not affected. NCB-0846 also reduces the expression of TNIK, AXIN2 and cMYC at the protein level. LRP6 and LRP5 are also downregulated by NCB-0846. NCB-0846 can inhibit cancer cell growth in vitro. NCB-0846 induces an increase in the sub-G1 cell population. NCB-0846 can downregulate the expression of putative colorectal CSC markers: CD44, CD133, and aldehyde dehydrogenase-1 (ALDH1), and reduce the proportion of cells showing high expression of CSC surface markers (CD44, CD133, CD166, CD29 and EpCAM). NCB-0846 also reduces the expression of mesenchymal markers (Slug, Snail, Twist, Smad2 and Vimentin). However, embryonal stem cell markers (Oct4, Nanog and Sox2) are not affected. In vivo: NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apcmin/+ mice and the sphere- and tumour-forming activities of colorectal cancer cells. The body weight of mice (immunodeficient tumor xenografts) falls at the beginning of NCB-0846 administration, but gradually recover. The expression of Wnt-target genes (AXIN2, MYC and CCND1) in xenografts is reduced following the administration of NCB-0846. NCB-0846 dose dependently reduces the multiplicity and dimensions of tumours that developed in the small intestine. NCB-0846 significantly suppresses the growth of the PDXs (patient-derived xenografts) established from the two patients in two more clinically relevant mouse models.

Protocol (for reference only)

Cell Experiment
Cell lines	HCT116 cells
Preparation method	HCT116 cells are cultured in the presence of DMSO (vehicle), 1 μM NCB-0846, or 1 μM NCB-0970 for 4 or 24 h and then analysed by immunoblotting with anti-phosphorylated TNIK, anti-TNIK, and anti-γ-tubulin (loading control) antibodies.
Concentrations	1 μM
Incubation time	4 or 24 h

Animal Experiment
Animal models	Immunodeficient mice (background-BALB/c nude mice)
Formulation	DMSO/polyethylene glycol#400/30% 2-hydroxypropyl-β-cyclodextrin solution (10:45:45, v/v)
Dosages	40 or 80 mg/kg BID
Administration	by oral gavage

Chemical Information

Molecular Weight	375.42
Formula	C21H21N5O2
CAS Number	1792999-26-8
Solubility (25°C)	65 mg/mL in DMSO
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Yamada T, et al. Cancer Sci. Emergence of TNIK inhibitors in cancer therapeutics.

[2] Masuda M, et al. Nat Commun. TNIK inhibition abrogates colorectal cancer stemness.