Discovery of a novel DNA polymerase inhibitor and characterization of its antiproliferative properties

Abstract

Chromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silico screening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces "BRCAness" in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

Keywords: DNA polymerase delta; DNA repair; DNA replication; PARP inhibitor; olaparib; polymerase inhibitor.

Figure 1.

Flow-chart of the in silico screening protocol by Dock 6.4 of the ChemoBridge library.

Figure 2.

Interactions of Zelpolib at the active site of Pol δ. A, Zelpolib forms two hydrogen bonds with dG on the template strand. The catechol moiety stacks against dOC (dideoxycytosine) on the primer. B, comparative position of dCTP at the active site of Pol δ. C, chemical structure of Zelpolib. D, 5 potential hydrogen-bonds can be formed between Zelpolib and Pol δ.

Figure 3.

Comparison of Zelpolib with dCTP in binding to the active site of Pol δ. Zelpolib in stick (A) and ball (B) representation with dCTP (C and D) in comparison.

Figure 4.

Zelpolb inhibits Pol δ activity. A, enzymatic assay by poly(dA)/oligo(dT) method demonstrates noncompetitive inhibition with K_i of 4.3 μM. B, Zelpolib is likely unique in inhibiting Pol δ activity in comparison to current FDA approved small molecule oncology drugs. C, quantification of the full-length products (40mer, integration by ImageJ). Lower peak height indicates less product. D, quantification of unextended primer. Higher peaks correspond to higher amounts of primer left.

Figure 5.

Zelpolib inhibits cellular DNA replication. A, EdU incorporation by whole cell population is inhibited by Zelpolib. Exponentially growing HCC1395 (TNBC) cells were treated with Zelpolib for 2 hours prior to pulse labeling with EdU for 30 minutes. Amount of EdU quantified with “click” chemistry and measured by laser scanning cytometry (LSC). Error bar shows mean value with SEM (triplicates) and P values were calculated using unpaired Student’s t-test. B, treatment scheme of DNA fiber fluorography assay. C, DNA fiber-length comparison between untreated and treated with 50 μM Zelpolib (HCC1395 cells, see Figure S4 for original images). D, quantification of DNA fiber length for BxPC-3 cells. E, quantification of DNA fiber length for HCC1395 cells. 75 fibers were analyzed per sample. Scattered dot plot shows ratio of green/red fiber lengths (ratios) with SEM. P values were calculated using unpaired T test. *** indicates p < 0.0001.

Figure 6.

Antiproliferative activities of Zelpolib. Concentration dependent inhibition of cell proliferation by Zelpolib on three different cell lines and comparison with methotrexate, 5-FU, and cisplatin by MTT assays. All samples were in triplicates and presented as averages with standard deviation.

Figure 7.

Zelpolib inhibits homologous recombination in cell-based assay. a, Dual plasmids (DR-GFP and I-Scel) reporter assay was used to measure the effect of Zelpolib on DSB repair in 293T cells as reported³⁴. The error bar represents mean values of four repeats with SEM. P values were calculated using unpaired T test. b, Zelpolib enhances the sensitivity of triple negative breast cancer cells (HCC1395) to niraparib by MTT assay. c, Zelpolib enhanced the sensitivity of TNB cells (HCC1395) towards Rucaparib.