Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25 and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively non-selective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4, KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine adjacent to the ATP binding site of TAK1.

Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1-dependent cell lines from 3 distinct cancer types.

Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691 showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1, ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells.

Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may have value as therapeutic agents and scientific tools.

Citation Format: Deepak Gurbani, Li Tan, Scott Ficarro, John C. Hunter, William Singer, Faviola B. Vazquez, Ting Xie, Sang Min Lim, Jarrod Marto, Nathanael S. Gray, Kenneth D. Westover. Structure guided development of irreversible inhibitors for TAK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A178.