Identification of a small molecule inhibitor of the human DNA repair enzyme polynucleotide kinase/phosphatase.
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.
Experimental Oncology, Cross Cancer Institute, and Departments of Oncology, Chemistry, and Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Gary K
GK
Freschauf
Feridoun
F
Karimi-Busheri
Agnieszka
A
Ulaczyk-Lesanko
Todd R
TR
Mereniuk
Ashley
A
Ahrens
Jonathan M
JM
Koshy
Aghdass
A
Rasouli-Nia
Phuwadet
P
Pasarj
Charles F B
CF
Holmes
Frauke
F
Rininsland
Dennis G
DG
Hall
Michael
M
Weinfeld
D000230:Adenocarcinoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D004260:DNA Repair; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D051379:Mice; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011116:Polynucleotide 5'-Hydroxyl-Kinase