Format

Send to

Choose Destination
DNA Repair (Amst). 2014 Sep;21:177-82. doi: 10.1016/j.dnarep.2014.03.006. Epub 2014 Apr 29.

Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput screening platform.

Author information

1
Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States.
2
National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Bethesda, MD 20892, United States.
3
National Clinical Target Validation Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States.
4
Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States; National Clinical Target Validation Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States.
5
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan.
6
Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States. Electronic address: pommier@nih.gov.

Abstract

Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1-/-) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1-/- cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1-/- cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors.

KEYWORDS:

Combination therapy; Drug discovery; PARP; TDP1; Topoisomerases

PMID:
24794403
PMCID:
PMC4125495
DOI:
10.1016/j.dnarep.2014.03.006
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center