Synergistic Effects of an Irreversible DNA Polymerase Inhibitor and DNA Damaging Agents on HeLa Cells

Abstract

DNA repair is vital to maintaining genome integrity, but thwarts the effects of cytotoxic agents that target nucleic acids. Consequently, repair enzymes are potential targets for molecules that modulate cell function and anti-cancer therapeutics. DNA polymerase β (Pol β) is an attractive target because it plays a key role in base excision repair (BER), a primary pathway that repairs the effects of many DNA damaging agents. We previously identified an irreversible inhibitor of Pol β whose design was based upon a DNA lesion that inactivates Pol β and its back up BER enzyme, DNA polymerase λ (Pol λ). Using this molecule as a starting point we characterized an irreversible inhibitor (13) of Pol β (IC50 = 0.4 μM) and Pol λ (IC50 = 1.0 μM) from a 130-member library of candidates that is ~50-fold more effective against Pol β. Pro-13 (5 μM) is only slightly cytotoxic to human cervical cancer cells (HeLa), but potentiates the cytotoxicity of methyl methanesulfonate (MMS). DNA isolated from HeLa cells treated with MMS contain a ~3-fold greater amount of abasic sites when pro-13 is present, consistent with inhibition of DNA repair. Proinhibitor pro-13 continues to induce cytotoxicity in DNA damaged cells following MMS removal. HeLa cell cytotoxicity is increased ~100-fold following a 8 h incubation with pro-13 after cells that were originally subjected to conditions under which 20% of the cells survive and reproduce. The potentiation of MMS cytotoxicity by pro-13 is greater than any previously reported BER enzyme repair inhibitor.

Translesion DNA Synthesis in Cancer: Molecular Mechanisms and Therapeutic Opportunities

“…Very recently, an irreversible inhibitor of pol β lyase activity was found to potentiate the cytotoxicity of MMS in HeLa cells through inhibition of abasic site repair. 141 A number of natural compounds have been shown to inhibit TLS pols in vitro and in some cases elicit antitumor responses in cell culture or animal models. For example, 3-Omethylfunicone, a compound isolated from an Australian fungal strain, was shown to inhibit pol κ with good selectivity.…”

Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities

“…The identification of a single nucleophilic residue within the active site of Pol θ suggests that a previously reported approach used for identifying Pol β inhibitors will be useful against this enzyme. 36…”

Base excision repair and its implications to cancer therapy

“…NSC666715 also appeared to potentiate the effects of TMZ in inducing cellular senescence in these cell lines. More recently, Pro-13, an irreversible inhibitor of Polβ (and Polλ) with an IC 50 of 0.4 µM demonstrated little cytotoxicity in HeLa cells, but had a large synergistic effect in combination with MMS [ 63 ]. Natamycin, an antibiotic/anti-fungal agent, has been shown to inhibit the strand displacement activity of Polβ (at 2–5 nM), and at higher (µM) concentrations inhibited both Polβ and LigI, and consequently reduced proliferation of androgen-depleted prostate cancer cell lines [ 64 ].…”