Structure-Based Design and Development of 2-Oxindole Derivatives as Next Generation Ku-DNA Binding Inhibitors (Ku-DBi’s) for Cancer Therapy.

The Ku70/Ku80 heterodimer (Ku) plays a key role in the non-homologous end joining (NHEJ) pathway by recognizing DNA double-strand breaks (DSBs) and recruiting DNA-PKcs to initiate DNA repair. Aberrant activation of this pathway contributes to therapy resistance in cancer by enhancing DNA repair capacity. Although DNA-PK inhibitors show strong in vitro potency, their clinical advancement has been hindered by poor selectivity, low tumor accumulation, and systemic toxicity, preventing the achievement of maximum tolerated doses. To address these limitations, we targeted the Ku-DNA interaction and were the first to develop Ku-DNA binding inhibitors (Ku-DBi’s) that block the Ku70/Ku80 interaction with DNA, thereby inhibiting DNA-PK activation and downstream repair. Our earlier Ku-DBi’s exhibited potent inhibitory activity and selectivity, but their in vivo utility was restricted by poor solubility, metabolic stability, and limited cellular permeability. In this study, we identified lead compounds with a novel 2-oxindole scaffold through iterative structure-activity relationship (SAR) optimization. This series of compounds maintained potent biochemical activity while exhibiting enhanced solubility, cellular permeability, and metabolic stability. Lead candidates 34 (GL-3395), 35 (GL-3392), 47 (GL-3367), and 53 (GL-3609) inhibited Ku-DNA binding at 1-4 µM and DNA-PK at 0.30-0.80 µM. Notably, compounds 19 (GL-3391), 35 (GL-3392), and 38 (GL-3366) exhibited significantly enhanced cellular uptake in both A549 and H460 cells under standard conditions without serum-free media. Molecular docking and in silico ADME profiling supported their favorable binding and drug-like properties. These findings offer a promising foundation for advancing Ku-DBi as an adjuvant therapy for DNA damage-based cancer treatment.