Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Abstract

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5 ¶-termini and dephosphorylates DNA 3 ¶-termini. hPNKP is involved in both single-and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to ;-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC 50 of 0.06 Mmol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3 ¶-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 Mmol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

Mechanism of Action of an Imidopiperidine Inhibitor of Human Polynucleotide Kinase/Phosphatase

“…5, compared with the positive control in the absence of inhibitor, there was no significant inhibition of incorporation of the missing nucleotide by Pol ␤ or joining of the two shorter oligonucleotides by DNA ligase III when acting on their respective substrates, even at the highest concentration of A12B4C3 tested (50 M). In contrast the human PNKP phosphatase activity is almost 100% inhibited by this concentration of A12B4C3 (14).…”

“…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 recently identified a series of compounds from a library of polysubstituted imidopiperidines that inhibit the 3Ј-phosphatase activity of PNKP (14). The most potent of these inhibitors, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo [3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3) (Fig.…”

“…The most potent of these inhibitors, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo [3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3) (Fig. 1), was shown to be non-toxic to A549 human lung cancer and MDA-MB-231 human breast cancer cells at 1 M, but increased the sensitivity of these cells to ionizing radiation to almost the same extent as stable shRNA-mediated PNKP depletion (14). On the other hand, A12B4C3 failed to enhance the sensitivity of the shRNA-mediated PNKP-depleted cells, indicating that PNKP is probably the major cellular target for the inhibitor.…”

Genetic Screening for Synthetic Lethal Partners of Polynucleotide Kinase/Phosphatase: Potential for Targeting SHP-1–Depleted Cancers

“…Given the potential to target PNKP by small molecule inhibitors (12), we sought to identify synthetic lethal relationships of PNKP to expand the repertoire of targeted therapy taking advantage of this approach. By screening approximately 7,000 genes targeting the "druggable" genome, we have identified a variety of proteins potentially synthetic lethal with PNKP including several that are either known or are implicated as tumor suppressors, such as the protein tyrosine phosphatase SHP-1 (PTPN6).…”

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates

“…The critical role of PNKP in various DNA repair processes is underlined by the fact that RNAi knockdown of PNKP leads to marked sensitization of human cells to spontaneous mutation as well as to various genotoxic agents (2). A particularly important role is attributed to the phosphatase activity as small molecules that selectively inhibit the phosphatase but not the kinase activity of PNKP sensitize human cells to DNA damage in a PNKP-dependent manner (3,4). Mutations in the PNKP phosphatase domain are associated with the developmental neurological disorder MCSZ, revealing a critical role for PNKP in human neurological development (5).…”