From phenotypic screening to mechanistic insight: Rational discovery of chemosensitizers for glioblastoma

Glioblastoma multiforme (GBM) represents the most aggressive form of primary brain tumor, characterized by rapid progression, therapeutic resistance, and near-universal recurrence. Despite multimodal treatment strategies-comprising maximal surgical resection followed by radiotherapy and temozolomide (TMZ)-based chemotherapy-median survival remains dismal, with most patients succumbing within one-year post-diagnosis. TMZ and ionizing radiation exert their cytotoxic effects primarily through the induction of DNA lesions, including single- and double-strand breaks. The cellular DNA damage response (DDR), encompassing a complex signaling network of kinases, repair enzymes, and checkpoint regulators, orchestrates the detection and repair of these lesions. Dysregulation or hyperactivation of DDR pathways-including those mediated by ATR/ATM signaling, PARP-dependent repair, and cell cycle checkpoints (e.g., WEE1, CDK1)-has been implicated in the pronounced therapeutic resistance of GBM. In this study, we pursued the rational design and synthesis of novel small molecules aimed at potentiating TMZ- and radiation-induced cytotoxicity in GBM. Over seventy compounds were generated across five focused series using structure-based design principles. Phenotypic screening was conducted in the TMZ-resistant T98G line and patient-derived GBM cultures to evaluate both intrinsic cytotoxicity and TMZ sensitization. Several lead candidates demonstrated superior chemosensitization efficacy compared to clinical DDR inhibitors. Interestingly, broad kinase profiling revealed an absence of canonical DDR kinase inhibition, indicating a potential non-classical mechanism of action. To delineate this mechanism, comprehensive quantitative proteomic analyses were performed, revealing modulation of pathways associated with DNA repair, stress response, and cell cycle control. The most promising candidates exhibited favorable pharmacokinetic and toxicological properties, along with effective blood-brain barrier penetration in vivo. This work has been supported by the grant from the Ministry of Health of the Czech Republic (NW24J-03-00005); and by the Long-term development plan Military Faculty of Medicine, Healthcare Challenges of WMD II (DZRO-VLF22-ZHN II).