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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2009-10, Vol.69 (19), p.7739-7746
• Main Authors: FRESCHAUF, Gary K, KARIMI-BUSHERI, Feridoun, HALL, Dennis G, WEINFELD, Michael, ULACZYK-LESANKO, Agnieszka, MERENIUK, Todd R, AHRENS, Ashley, KOSHY, Jonathan M, RASOULI-NIA, Aghdass, PASARJ, Phuwadet, HOLMES, Charles F. B, RININSLAND, Frauke
• Format: Article
• Language: English
• Subjects: Adenocarcinoma - drug therapy; Adenocarcinoma - enzymology; Adenocarcinoma - genetics; Animals; Antineoplastic agents; Biological and medical sciences; Breast Neoplasms - drug therapy; Breast Neoplasms - enzymology; Breast Neoplasms - genetics; Cell Line, Tumor; DNA Repair; Enzyme Inhibitors - pharmacology; Humans; Lung Neoplasms - drug therapy; Lung Neoplasms - enzymology; Lung Neoplasms - genetics; Medical sciences; Mice; Pharmacology. Drug treatments; Polynucleotide 5'-Hydroxyl-Kinase - antagonists & inhibitors; Substrate Specificity; Tumors
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-09-1805

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 gamma-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 IC50 of 0.06 micromol/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 micromol/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.