Abstract 3235: Identification of the potential inhibitors of DNA polymerase kappa

Abstract

Abstract Background and Purpose: The therapeutic effect of many anticancer agents is due to induction of replication-blocking DNA lesions. Human DNA polymerase kappa (pol κ) is a trans-lesion synthesis polymerase that can catalyze direct replication past minor groove lesions, such as the one induced by mitomycin C. Pol κ has been found to be upregulated in ∼70% of gliomas and its upregulation associated with poorer prognosis in glioma patients. Therefore, small molecule inhibitors of pol κ in combination with chemotherapy may be useful in the treatment of cancer with upregulated pol κ expression. Methodology: Lead hits were initially identified from a screen of a library of ∼400,000 compounds at 5 different concentrations, using a strand-displacement DNA synthesis approach. From several thousand potential hits, these were systematically triaged, with 335 potential inhibitors of pol κ identified for further analyses. A direct, gel-based primer extension assay was used to test for inhibitory effects of these 335 bioactive compounds. In this assay, pol κ was preincubated with individual compounds, and added to 32P-labeled DNA substrate to initiate primer extensions. The reactions were incubated for 30 minutes at room temperature and terminated by the addition of a DNA-denaturing solution. The products were then separated through a 15% acrylamide gel and visualized with a PhosphorImager screen. Compounds that significantly inhibited the polymerization activity of pol κ were further tested for the ability to intercalate into DNA by incubation with double-stranded DNA for 15 minutes at room temperature, followed by separation through a 1% agarose gel. DNA was stained with ethidium bromide and visualized by Alpha Innotech ChemiImager. Results: Through a series of rigorous triaging procedures, this investigation identified a total of 42 small molecule inhibitors of pol κ with the estimated potencies being the low micromolar range or less. All 42 compounds were confirmed not to exhibit their inhibitory effects through DNA intercalation. These compounds need to be further tested with other members of the Y family polymerase to ensure their specificity to pol κ and determine if they are amenable to medicinal chemistry. Conclusion: Further investigation and characterization of these compounds will allow for a more efficient therapeutic approach in patients who have upregulated pol κ receiving chemotherapy. Citation Format: Christopher B. Sullivan, Irina G. Minko, Kinrin Yamanaka, Amanda K. McCullough, Stephen Lloyd. Identification of the potential inhibitors of DNA polymerase kappa. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3235. doi:10.1158/1538-7445.AM2014-3235