Development of phenothiazine-based inhibitors towards drug-resistant glioblastoma

Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy characterized by extensive genomic instability and poor response to existing therapies. Emerging evidence suggests that threonine tyrosine kinase (TTK/Mps1) plays an important role in clonogenicity and tumor progression in GBMs. Herein, we designed and synthesized a series of phenothiazine-based TTK inhibitors and evaluated their anti-GBM activity. Biological studies identified G17b as the most effective analog, exhibiting concentration-dependent TTK inhibitory activity together with significant antiproliferative effects in LN229 and U87MG cells. Importantly, G17b retained substantial cytotoxic activity in TMZ-resistant LN229 cells under conditions where TMZ, a DNA-alkylating agent acting through a distinct mechanism, showed minimal efficacy. Mechanistic investigations further demonstrated that G17b impaired migratory capacity, suppressed clonogenic survival, induced intracellular ROS accumulation, and promoted apoptotic cell death in both TMZ-sensitive and -resistant GBM models. Collectively, these findings highlight phenothiazine-based TTK inhibition as a promising therapeutic strategy for drug-resistant glioblastoma.