Comprehensive Molecular Dynamics Simulation and Immunoblotting Analysis of Novel Azole Compound as Potential Inhibitor of TRAF2 and NCK-Interacting Kinase (TNIK) in Colorectal Cancer

Colorectal Cancer (CRC) is one of the most prominent cancer-causing mortalities globally, which is primarily influenced by the dysregulation of the Wnt/β-catenin signalling pathway. TRAF2- and NCK-interacting kinase (TNIK) is a crucial regulator involved in this pathway, functioning by phosphorylating the transcription factor TCF4 to maintain the oncogenic gene expression. In our study, we investigated the effectiveness of synthetic azole derivatives as TNIK inhibitors using in silico and in vitro analysis. Among the three evaluated azole derivatives, LC_112060 exhibited the most potent anticancer activity and subsequent mechanistic investigations, including gene and protein expression analyses, molecular docking, molecular dynamics simulations, principal component analysis, free energy landscape analysis, and residue interaction network studies.The semi-quantitative RT-PCR and immunoblotting analyses demonstrated the specificity of LC_112060 for TNIK, further downregulating TNIK and TCF mRNA and protein expression, leading to disruption of the β-catenin/TCF4 transcriptional complex. Computational docking revealed a high binding affinity of LC_112060 for the TNIK ATP-binding pocket, exceeding that of ATP and the reference inhibitor mebendazole. Molecular dynamics simulations corroborated that LC_112060 stabilises TNIK in fewer, low-energy conformational states, with constrained RMSD and a compact radius of gyration, while residence-time analysis revealed prolonged occupancy of the binding pocket. Principal component analysis, free energy landscape mapping, and elastic network modelling consistently demonstrated that LC_112060 constrains TNIK's conformational flexibility. These findings collectively underscore LC_112060 as a potent and selective TNIK inhibitor that may negatively affect Wnt/β-catenin signalling and induce cell death in colorectal cancer (CRC). Given the TNIK's growing significance across various oncogenic pathways, the compound LC_112060 is a promising lead candidate for subsequent preclinical development in targeted CRC therapy.