Identification of dimethyl 2,2'-((methylenebis(2-(2H-benzo[d][1,2,3]triazol-2-yl)-4-(2,4,4-trimethylpentan-2-yl)-6,1phenylene))bis(oxy))diacetate (TAJ4) as antagonist of NEK-Family: a future for potential drug discovery.

The purpose of the current study was to analyze and validate the existing gap in knowledge, by conducting a differential expression analysis and validation of NEK6, NEK7, and NEK9 in breast, cervical, and glioblastoma cancer and targeting these proteins through development of novel site specific inhibitor with favorable pharmacokinetic and safety profile, using open-source databases. The analysis revealed that the targeted kinases were overexpressed in all three types of cancer. Their expression was significantly linked to overall survival rates, which suggests that they play a major role in the development and progression of these cancers. After, having the prognostic importance of These findings provided a rationale for synthesizing novel compound i.e., dimethyl 2,2'-((methylenebis(2-(2H-benzo[d][1,2,3]triazol-2-yl)-4-(2,4,4-trimethylpentan-2-yl)-6,1phenylene))bis(oxy))diacetate (TAJ4)), capable of effectively targeting these proteins using in-vitro cytotoxicity assays and comprehensive computational approaches. Then the inhibitory potential of TAJ4 was evaluated against cell lines of the respective cancers (HeLa cells, MCF-7 cells, and Vero cells). The growth inhibitory values (GI) suggested that TAJ4 exhibited strong inhibitory potential towards MCF-7 cells (GI = 3.18 ± 0.11 µM) in comparison to the HeLa cell line (GI = 8.12 ± 0.43 µM), surpassing that of standard drugs. Furthermore, in-silico investigations, including density functional theory (DFT) calculations and molecular docking studies, revealed a substantial reactivity profile of TAJ4, with promising molecular interactions against NEK7, NEK9, TP53, NF-KAPPA-B, and caspase-3 proteins. Further investigation using in-vitro and in-vivo approaches is recommended to fully establish the therapeutic efficacy and safety profile of TAJ4.