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

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.

Figure 1

Screening strategy. A: A quantitative robotic high throughput screening (qHTS) assay was run as a primary screen using DT40 chicken B lymphoma cells genetically modified to express human TDP1 (hTDP1) in a knockout background for the chicken TDP1 gene (TDP1-/-) [23]. The compound library was the NIH Roadmap Molecular Libraries 400,000 compound repository. Positive hits were selected based on cellular hypersensitivity in the presence of the Top1 inhibitor camptothecin (CPT). B: Positive hits identified during the HTS assay were confirmed in a secondary screen against both hTDP1 and TDP1-/- cells. Inhibitors of the TDP1 pathway were selected for further characterization based on supra-additive cytotoxicity in the presence of CPT in hTDP1 cells but not in TDP1-/- cells.

Figure 2

Inhibition of PARP1 by compound 1 (Cpd1). A & B: Cellular viability in the presence of Cpd1 and various concentrations of CPT (indicated beside each curve in nanomolar unit) in hTDP1 (A) and TDP1-/- cells (B). C & D: Cellular viability curves in the presence of veliparib and various concentrations of CPT (indicated beside each curve in nanomolar unit) in hTDP1 (C) and TDP1-/- cells (D). E & F: Inhibition of PARP1 by compounds 1-5 in comparison to the PARP inhibitor veliparib measured by ELISA (E) and by Western blotting (F). Asterisks in (F) indicate non-specific bands. G: Cpd1-dependent viability curves in PARP1-/-, BRCA2tr/- and hTDP1 cells.