title: A novel small molecule inhibitor of the DNA repair protein Ku70/80.
abstract: Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.
authors.affiliation: Department of Radiation Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States. Electronic address: eweterings@email.arizona.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Weterings
authors.affiliation: Department of Radiation Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States.
authors.forename: Alfred C
authors.initials: AC
authors.lastname: Gallegos
authors.affiliation: Department of Radiation Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States.
authors.forename: Lauren N
authors.initials: LN
authors.lastname: Dominick
authors.affiliation: Division of Hematology/Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States.
authors.forename: Laurence S
authors.initials: LS
authors.lastname: Cooke
authors.affiliation: Department of Radiation Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States.
authors.forename: Trace N
authors.initials: TN
authors.lastname: Bartels
authors.affiliation: Bio5 Institute, Ligand Discovery Lab, University of Arizona, 1657 E. Helen St, Tucson, AZ 85721, United States.
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
authors.affiliation: Department of Medical Imaging, University of Arizona, 1609N. Warren Street, building 211, Tucson, AZ 85724, United States.
authors.forename: Terry O
authors.initials: TO
authors.lastname: Matsunaga
authors.affiliation: Division of Hematology/Oncology, University of Arizona Cancer Center, 1515N. Campbell Ave, Tucson, AZ 85724, United States.
authors.forename: Daruka
authors.initials: D
authors.lastname: Mahadevan
mesh_terms: D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000187:drug effects*; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005720:Gamma Rays; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011743:Pyrimidines / Q000138:chemical synthesis / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000494:pharmacology*
chemical_list: D009687:Nuclear Proteins; D011743:Pyrimidines; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen
keywords: Chemotherapy; DNA double strand break; Ku70/80; Non-Homologous End-Joining; Radiation; Small molecule inhibitor