Novel 5,7-Diazaindole-based ERK5 inhibitor induces endoplasmic reticulum stress and mitochondrial apoptosis in non-small cell lung Cancer.

The extracellular signal-regulated kinase 5 (ERK5) signaling pathway represents a promising therapeutic target for non-small cell lung cancer (NSCLC), yet the development of potent and selective inhibitors remains a challenge. Leveraging the 5,7-diazaindole scaffold, a privileged structure in kinase inhibitor discovery, we designed, synthesized, and evaluated a novel series of derivatives as potential ERK5 inhibitors. Among them, compound I1 emerged as the most potent candidate, demonstrating significant anti-proliferative activity against A549 human lung cancer cells with an IC₅₀ of 40.1 μM. Critically, an in vitro kinase assay confirmed that I1 is a direct ERK5 inhibitor, exhibiting potent inhibition of purified ERK5 kinase activity with an IC₅₀ of 403.4 nM. Structure-activity relationship (SAR) studies underscored the critical importance of the unsaturated 1,2,3,6-tetrahydropyridine ring, the amide carbonyl group, and the N1H moiety for optimal activity. Molecular docking revealed that I1 binds robustly within the ERK5 ATP-binding site (PDB: 6HKM), forming key hydrogen bonds with Met140, Asp138, and Asp200, and exhibiting a more favorable binding mode than its analogues. Mechanistic studies indicated that I1 functions as a direct ERK5 inhibitor, suppressing both ERK5 phosphorylation and total protein expression. This ERK5 inhibition triggered a multi-modal anti-tumor mechanism, including the induction of endoplasmic reticulum stress, mitochondrial dysfunction (characterized by reactive oxygen species accumulation and loss of membrane potential), and ultimately, activation of the mitochondrial apoptotic pathway. Importantly, I1 exhibited significant dose-dependent tumor growth suppression in a Lewis lung carcinoma mouse model without causing observable toxicity, highlighting its potential as a promising lead compound for targeted NSCLC therapy.