Abstract
Base excision repair (BER) is a DNA repair pathway responsible for protecting the genome against modified nucleotides. DNA polymerase β (Pol β) participates in this process by removing the remnants of a damaged nucleotide and filling in the resulting gap. Pol β is overexpressed in some cancers and is synthetic lethal in cells deficient in BRCA1/2, providing additional impetus for identifying inhibitors of this enzyme. We report noncovalent Pol β inhibitors that are nonnative nucleotides. The inhibitors were identified via a combination of structural and biochemical analysis, as well as serendipity, from an initial library of covalent inhibitor candidates in which diversity was introduced sequentially at the C3'- and C5-positions of pyrimidine nucleotides. The molecules are among the most potent Pol β inhibitors (K(i) ≤ 70 nM) of the enzyme's polymerase and lyase activities. Kinetic analyses reveal that the molecules inhibit Pol β noncompetitively. Fluorescence anisotropy and kinetic experiments reveal that the more potent inhibitor binds in the lyase domain and does not prevent DNA binding. Neither the more potent noncompetitive inhibitor nor a neutral protide exhibits cytotoxic synergism with the DNA damaging agent methyl methanesulfonate in HeLa cells. Cell permeability experiments suggest that micromolar levels of the more potent noncompetitive inhibitor and corresponding protide are taken up by HeLa cells following 24 h incubation (25 μM). However, based upon a comparison with other molecules, it is possible that they are membrane bound. The molecules identified could be useful tools in biochemical studies and provide a starting point for creating new Pol β inhibitors that function in cells.