Abstract
The small molecule, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), is a potent inhibitor of the phosphatase activity of human polynucleotide kinase/phosphatase (PNKP) in vitro. Kinetic analysis revealed that A12B4C3 acts as a noncompetitive inhibitor, and this was confirmed by fluorescence quenching, which showed that the inhibitor can form a ternary complex with PNKP and a DNA substrate, i.e. A12B4C3 does not prevent DNA from binding to the phosphatase DNA binding site. Conformational analysis using circular dichroism, UV difference spectroscopy, and fluorescence resonance energy transfer all indicate that A12B4C3 disrupts the secondary structure of PNKP. Investigation of the potential site of binding of A12B4C3 to PNKP using site-directed mutagenesis pointed to interaction between Trp(402) of PNKP and the inhibitor. Cellular studies revealed that A12B4C3 sensitizes A549 human lung cancer cells to the topoisomerase I poison, camptothecin, but not the topoisomerase II poison, etoposide, in a manner similar to small interfering RNA against PNKP. A12B4C3 also inhibits the repair of DNA single and double strand breaks following exposure of cells to ionizing radiation, but does not inhibit two other key strand-break repair enzymes, DNA polymerase beta or DNA ligase III, providing additional evidence that PNKP is the cellular target of the inhibitor.
Highlights
We previously identified A12B4C3 from a library of polysubstituted imidopiperidines based on its ability to inhibit the phosphatase activity of polynucleotide kinase/phosphatase (PNKP) in vitro [14]
To confirm this, we further compared the properties of A12B4C3-treated cells to A549␦PNKP cells, in particular their response to topoisomerase I and II poisons, camptothecin and etoposide
The 3Ј-phosphate and 5Ј-hydroxyl strand-break termini generated in camptothecintreated cells require the remedial action of PNKP, unlike the termini generated by etoposide
Summary
Cancer cells in which PNKP expression has been depleted by targeted shRNA display a noticeable decrease in cell survival when exposed to a number of genotoxic agents, including ionizing radiation, camptothecin, and the alkylating agent, methyl methanesulfonate [4]. This suggests that PNKP plays an important role in countering exogenous DNA damage, and it was proposed that PNKP may be a suitable target for small molecule inhibitors to enhance the toxicity of genotoxic therapeutic agents, especially ionizing radiation and topoisomerase I poisons [12, 13]. We have obtained information regarding the perturbation of PNKP by A12B4C3 and the site of interaction of the protein with the inhibitor
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