TY - JOUR
T1 - A Comprehensive Strategy to Discover Inhibitors of the Translesion Synthesis DNA Polymerase κ
AU - Yamanaka, Kinrin
AU - Dorjsuren, Dorjbal
AU - Eoff, Robert L.
AU - Egli, Martin
AU - Maloney, David J.
AU - Jadhav, Ajit
AU - Simeonov, Anton
AU - Lloyd, R. Stephen
PY - 2012/10/8
Y1 - 2012/10/8
N2 - Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N2-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.
AB - Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N2-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.
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U2 - 10.1371/journal.pone.0045032
DO - 10.1371/journal.pone.0045032
M3 - Article
C2 - 23056190
AN - SCOPUS:84867304060
SN - 1932-6203
VL - 7
JO - PloS one
JF - PloS one
IS - 10
M1 - e45032
ER -