In vitro activity: NCB-0846 is a novel, first orally bioavailable small molecule inhibitor of Wnt that inhibits TNIK (TRAF2 and NCK-Interacting Kinase) with IC50 value of 21 nM. 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, therefore the inhibition of TNIK by NCB-0846 may be a promising therapeutic approach.

Kinase Assay: 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.

Cell Assay: HEK293 cells were transiently transfected with pGL4.49[luc2P/TCF-LEF-RE/Hygro] reporter vector (Promega) using Fugene HD Transfection Reagent. After overnight incubation, the cells were then treated with 10 mM LiCl (Sigma-Aldrich). Luciferase activity was measured using the ONE-Glo Luciferase Assay System (Promega). The assay was performed in triplicate and repeated at least two times.

Patient-derived colorectal cancer spheroids were dissociated, resuspended in medium containing 50% Matrigel (Corning), and inoculated subcutaneously (#6; 1 × 104 cells per injection, #19; 1 × 105 cells per injection) into the flank of female NOD/ShiJic-scid mice (4-week-old females; CLEA Japan). When the tumour volume reached ~200 mm3, the mice were randomized according tumour volume (10 mice per group) and treatment was started. NCB-1026 (hydrochloride salt of NCB-0846) was dissolved in sterile saline solution and administered BID by oral gavage at 0 (vehicle alone), 50 or 100 mg kg–1 (body weight) on a 6-days-on, 1-day-off schedule.

Patient-derived colorectal cancer xenografts COX021 and COX021 were established by Shanghai ChemPartner. Female Nu/Nu nude mice aged 5–6 weeks were implanted subcutaneously with the xenografts and randomized into three treatment groups (10 mice per group) based on the developed tumour volume (~200 mm3). NCB-1026 (hydrochloride salt of NCB-0846) dissolved in sterile water was administered BID by oral gavage at 0 (vehicle alone), 45, or 90 mg kg–1 (body weight) on a 5-days-on, 2-day-off schedule.