A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase κ.
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.
Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon, United States of America.
Kinrin
K
Yamanaka
Dorjbal
D
Dorjsuren
Robert L
RL
Eoff
Martin
M
Egli
David J
DJ
Maloney
Ajit
A
Jadhav
Anton
A
Simeonov
R Stephen
RS
Lloyd
D000171:Acrolein / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries; D013729:Terpenes / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology; D014466:Ultraviolet Rays; D002722:Chlorobenzenes; D000099270:Y-Family DNA Polymerases
D000171:Acrolein; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D004247:DNA; D018736:DNA Adducts; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D004791:Enzyme Inhibitors; D007211:Indoles; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries; D013729:Terpenes; D013777:Tetrazoles; C488128:alpha-hydroxypropanodeoxyguanosine; C060893:MK-886; D002722:Chlorobenzenes; C045673:manoalide; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C077793:candesartan cilexetil