Development of novel Ku-targeted DNA-PK inhibitors for cancer therapy

The DNA-dependent protein kinase (DNA-PK) is a clinically validated target for cancer therapy. It plays essential roles in the repair of DNA double-strand breaks (DSBs) via non-homologous end joining (NHEJ), and in the broader DNA damage response (DDR) signaling pathway. Given its pivotal roles in maintaining genomic stability, and its upregulation and hyperactivation in many cancers, DNA-PK is an attractive therapeutic target for single agent therapy and in combination with radiation or other DNA damaging chemotherapeutics. Existing DNA-PK inhibitors target the catalytic subunit (DNA-PKcs) but have suffered from poor selectivity, suboptimal pharmacokinetics, and dose-limiting toxicity. To overcome these limitations, we have developed a first-in-class series of small-molecule inhibitors that block Ku70/80 binding to DNA, the initial and essential step of DNA-PK activation. Our structure-based drug discovery program has led to the development of novel, drug-like Ku inhibitors that bind to unique pockets within the Ku interface, blocking DNA access and effectively shutting down DNA-PK activity. This novel, ATP-independent mechanism prevents assembly of NHEJ complexes, offering enhanced selectivity, reduced off-target toxicity, and a powerful new strategy for cancer therapy. Lead compounds potently inhibit DNA-PK catalytic activity at nanomolar concentrations, block Ku-dependent DNA binding, and sensitize cancer cells to ionizing radiation, etoposide, and bleomycin while sparing normal cells. Proof-of-concept studies confirm on-target mechanism, pharmacodynamic engagement, and enhancement of IR-induced antitumor activity in non-small cell lung cancer (NSCLC) xenograft models. Additionally, newly emerging and growing class of ADCs that induce DNA damage as their primary efficacious mechanism have been increasingly popular in the clinic. Ku inhibitors have strong mechanistic rationale as combination partners for ADCs, with the potential to overcome resistance and improve the therapeutic index of DNA damage inducing ADCs. Our results show significant increased efficacy in combination with the ADC topoisomeraseI inhibitor, DXd, suggesting that Ku inhibition enhances DXd-induced cytotoxicity by impairing non-homologous end joining and sensitizing tumor cells to payload-mediated DNA damage. These results support the further development of Ku inhibitors as anticancer therapeutics.