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

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.

Potentiation of hypericin-mediated photodynamic therapy cytotoxicity by MK-886: Focus on ABC transporters, GDF-15 and redox status

“…MK-886 does not increase ROS production after HY-PDT in HT-29 cells, but modulates their composition [19]. Taken together with previously published results [19,20,40] other treatments were described, including mitogen-activated protein kinase p38 signalization [42], increasing levels of death receptor [22], inhibition of DNA synthesis by error prone polymerases [68] and changes in proteins of Bcl family were also involved in MK-886 induced apoptosis [18]. These data could present an additional inspiration for our future experiments that will focus primarily on MK-886 influence on transporter proteins and its potential contribution to increase anticancer efficacy of their substrates.…”

N-Aroyl Indole Thiobarbituric Acids as Inhibitors of DNA Repair and Replication Stress Response Polymerases

“…33, 40 The assay relies upon polymerase-catalyzed displacement of a fluorescently-labeled oligonucleotide and shows excellent reproducibility (Figure 1B). Our initial screen to identify inhibitors of hpol η was performed with a final concentration of 6 μM for each compound.…”

“…31, 32 These specialized polymerases exhibit unique structural and functional properties that allow for the successful copying of DNA adducts, but these features also make them targets for small-molecule inhibitors. 33, 34 The mis-regulation and mutation of Y-family pols has been observed in many tumor types. 16, 35-39 Importantly, recent studies have shown that Y-family polymerases, particularly human DNA polymerase eta (hpol η), participate in mechanisms that promote resistance to anti-cancer treatments, such as cisplatin and doxorubicin.…”

DNA repair mechanisms and their clinical impact in glioblastoma

“…Recognition of DSBs by either KU70/KU80 or PARP leads respectively, to canonical NHEJ and alternative NHEJ (A-NHEJ, itself composed of sub-pathways), a backup pathway that has been described in GBM cells(24,25). Finally, translesion synthesis polymerases (not represented) that allow bypass of TMZ-induced lesions during DNA replication have been implicated as a mechanism to tolerate TMZ-induced DNA lesions(26)(27)(28).<Figure>Fig. 2.…”