Abstract
Human DNA polymerase kappa (pol κ) 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 κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ 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 ∼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 κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.
Highlights
Cells employ multiple mechanisms to repair or tolerate DNA lesions in order to maintain genomic integrity
Compounds with inhibitory activity against pol k have been identified, most of these inhibitors were discovered as part of screens of natural products against any DNA polymerase and their therapeutic potential may be limited primarily due to either low potency or poor selectivity
Pol k activity is inhibited by C12:0-Acyl juglone and C18:1Acyl juglone with IC50s of 6.8 mM and 8.1 mM, respectively [32]
Summary
Cells employ multiple mechanisms to repair or tolerate DNA lesions in order to maintain genomic integrity. Many clinically relevant chemotherapeutic agents, including mitomycin C, cisplatin, and nitrogen mustard, target tumor cells by virtue of their ability to covalently cross-link complementary DNA strands, introducing ICLs into the genome. TLS is an essential process for cells to survive genotoxic stress, the ability of pol k to bypass ICLs could limit the efficacy of these agents Critical to this point are data demonstrating that the effectiveness of mitomycin C was increased when pol k expression was suppressed by siRNA [12]. To the best of our knowledge, only one selective small molecule inhibitor of pol k has been identified to date: a natural product, 3O-methylfunicone [24] This compound exhibits selectivity against Y-family DNA polymerases, and importantly, among the Y-family polymerases investigated, it shows the highest potency towards pol k at IC50 of 12.5 mM. We report the new strategies to identify small molecule inhibitors of pol k
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