A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ

Human DNA polymerase kappa (pol kappa) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N-2-dG linked acrolein-and polycyclic aromatic hydrocarbon-derived adducts, as well as N-2-dG DNA-DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol kappa TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol kappa, small molecule library screens targeting pol kappa were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen similar to 16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol kappa by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of gamma-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol kappa. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol kappa, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol kappa inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.