Abstract Purpose: Non-Homologous End-Joining (NHEJ) is the primary pathway for the repair of DNA double strand breaks (DSBs) in human and mammalian cells. NHEJ is frequently upregulated in diverse solid malignancies and is a determining factor in the process of carcinogenesis. In addition, upregulation of the NHEJ pathway can reduce the efficiency of anti-cancer therapies including radiation or chemotherapy. Inhibition of NHEJ by siRNA reduces the growth of NHEJ-upregulated tumors and alleviates radiation-refractory disease. The Ku70/80 heterodimer is the central scaffolding protein of the NHEJ pathway, which recruits and activates all other key NHEJ factors. Therefore, Ku70/80 protein is a logical target for disruption of the NHEJ process. Experimental Procedures: We report the identification of a prospective binding pocket in the Ku70/80 crystal structure, located in close proximity to the DNA-binding domain and the Ku70/80 heterodimer interface. Based on this pocket, an idealized virtual ligand (‘protomol’) was created and utilized to screen ‘in silico’ an extensive database of small molecule compounds for potential ligands. Nine of the highest ranking hits were chosen for biological evaluation. Results: We identified one compound (designated ‘Compound L’), which proved capable of disrupting the in vitro binding of Ku70/80 to a DNA substrate in a dose-dependent manner with an IC50 of 3.5 μM. In addition, Compound L showed dose-dependent inhibition of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKCS) activity, which requires Ku70/80 as a co-factor with an IC50 of 2.5 μM. Finally, we showed that Compound L synergistically sensitizes a glioblastoma cell line to ionizing radiation, with a marked sensitization at ∼20-25 μM. This latter observation clearly demonstrates the ability of Compound L to disrupt NHEJ-mediated repair of radiation-induced DSBs. Conclusions: Compound L is an active inhibitor of the Ku70/80 heterodimer. To the best of our knowledge, no such compound has been reported in the peer-reviewed literature. The structure of Compound L will be utilized as a scaffold for a ‘structure-activity relationship’ (SAR) based hit-to-lead drug development aimed at designing a novel class of anti-cancer agents targeting the Ku70/80 heterodimer. We predict Ku70/80 inhibitors would have applicability as single-modality drugs, following a synthetic lethality approach, or as sensitizing agents for concurrent radiation or chemotherapy in a variety of solid malignancies. Support: P30 CA023074. Citation Format: Daruka Mahadevan, Alfred C. Gallegos, Lauren N. Dominick, Laurence S. Cooke, Trace N. Bartels, Josef Vagner, Terry O. Matsunaga, Eric Weterings. A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2749.