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Identification of novel PARP inhibitors using a cell-based TDP1 inhibitory assay in a quantitative high-throughput
Junko Murai1, Christophe Marchand1, Sampada A Shahane2
1Developmental Therapeutics Branch, Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United States.
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.
Insights
Researchers screened compounds to find new anti-cancer drugs. They discovered five compounds that enhance the effect of topoisomerase I inhibitors by blocking poly(ADP-ribose)polymerase (PARP) instead of TDP1, offering a new drug development avenue.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Topoisomerase I (Top1) inhibitors like camptothecin (CPT) create lethal DNA lesions by stabilizing the Top1-DNA cleavage complex (Top1cc).
- Repair pathways, including tyrosyl-DNA phosphodiesterase 1 (TDP1) and homologous recombination, handle these lesions.
- TDP1 deficiency increases sensitivity to Top1 inhibitors, suggesting TDP1 inhibition could enhance anti-cancer therapy.
Purpose of the Study:
- To discover novel inhibitors of the TDP1 pathway using a high-throughput screening assay.
- To identify compounds that synergize with camptothecin (CPT) to enhance anti-cancer cytotoxicity.
- To investigate the mechanism of action for identified synergistic compounds.
Main Methods:
- Developed a cell-based high-throughput screening assay using human TDP1 (hTDP1)-complemented and TDP1-deficient (TDP1-/-) chicken DT40 cells.
- Screened a 400,000-compound library for synergistic effects with CPT in hTDP1 cells.
- Confirmed hits in secondary screens and assessed direct TDP1 inhibition in vitro, followed by poly(ADP-ribose)polymerase (PARP) inhibition assays (ELISA, Western blotting).
Main Results:
- Identified five compounds showing synergistic effects with CPT in hTDP1 cells but not TDP1-/- cells, indicating inhibition of a TDP1-related pathway.
- In vitro assays revealed these compounds do not directly inhibit TDP1 catalytic activity.
- All five compounds were found to inhibit poly(ADP-ribose)polymerase (PARP) catalytic activity; the most potent compound (Cpd1) showed characteristics similar to the clinical PARP inhibitor veliparib.
Conclusions:
- The identified compounds potentiate CPT cytotoxicity by inhibiting PARP, not TDP1 directly.
- PARP inhibition likely enhances CPT efficacy by affecting Top1cc repair pathways.
- The novel compound Cpd1 represents a potential new scaffold for developing PARP inhibitors as anti-cancer therapeutics.
