Abstract B265: Targeting Wnt signaling: Discovery and characterization of novel thiazole-based Traf2- and NCK-interacting kinase (TNIK) inhibitors.

Abstract Introduction: Aberrant activation of Wnt pathway is a major force driving colorectal carcinogenesis, and therefore the Wnt pathway has been thought of as an attractive anticancer drug target. However, its complexity has made it difficult to find druggable targets in the Wnt pathway. Recently, we reported Traf2- and NCK-interacting kinase (TNIK) plays important roles in the Wnt signaling pathway by making a complex with beta-catenin and T-cell factor-4 (TCF4) in a nucleus. We also found that TNIK is activated in human colorectal cancer cell lines, and proliferations of those cells are highly dependent upon the expression and catalytic activity of TNIK. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating colorectal cancer. Here we describe a discovery and characterization of a novel TNIK inhibitor. Methods: To identify compounds that inhibit TNIK acitivity, we have produced a recombinant catalytic domain of human TNIK, and developed a high throughput screening (HTS) assay system using a peptide substrate. TCF/LEF reporter gene assay using HEK293 was established to validate hit compounds. To study the effects of TNIK inhbitors on the Wnt signal pathway, Wnt target genes were analyzed by RT-PCR and Western blotting. The selected compounds were subjected to a kinase selectivity panel assay. Results: By performing a high throughput screening campaigns of our 10K kinase-focused compound library against a recombinant human TNIK, we identified a series of novel thiazole compounds as a potent inhibitor of TNIK. Hit compound, NCB-0001 exhibited potent inhibitory activity for TNIK with an IC50 value of 8.6 nM. After subsequent optimization, N1662 was identified as a lead compound having a strong anti-proliferative activity in colorectal cancer cell lines. N1662 inhibited tumor growth in a mouse xenograft model of colorectal cancer. Further optimization led to compound N5355 that showed good TNIK enzyme inhibition and potent Wnt signal inhibition in the TCF reporter gene assay, as well as down-regulation of Wnt target genes. N5355 showed excellent kinase selectivity against a panel of 311 kinases. Detailed results will be presented in the conference. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B265. Citation Format: Yuko Uno, Hideki Moriyama, Shigeki Kashimoto, Yusuke Kawase, Miki Shitashige, Masaaki Sawa, Tesshi Yamada. Targeting Wnt signaling: Discovery and characterization of novel thiazole-based Traf2- and NCK-interacting kinase (TNIK) inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B265.