Novel Oxidatively Activated Agents Modify DNA and Are Enhanced by Ercc1 Silencing

Abstract

Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq2, which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and is found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed the agents add to 2′-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2′-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2′-deoxyadenosine and 2′-deoxycytidine, but not thymidine or 2′-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq2 compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between two and four fold above controls. Benzetheno-nucleobase addition to double stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi based approach, Ercc1 was silenced and survival monitored after injection of an agent. As expected, bulky cross-linking DNA modifying agents, cisplatin and chlorambucil, showed statistically significant enhanced toxicity in Drosophila with silenced Ercc1. In addition, 5-fluorouracil, which does not produce bulky lesions, showed no selective toxicity. An-Hq and An-Hq2 showed statistically significant toxicity in Drosophila with silenced Ercc1. Examination of cytotoxicity shows renal carcinoma cell lines as a target of these agents with a median IC50 of 1.8 μM. Taken together, this data shows that the designed oxidatively-activated agents form distinct, bulky DNA modifications that prove difficult for cancer cells possessing an elevated reactive oxygen species phenotype to overcome. The modification produced is relatively unique among anticancer agents.

A ROS‐Activatable Agent Elicits Homologous Recombination DNA Repair and Synergizes with Pathway Compounds

“…Despite this, modification of DNA occurred as shown through induction of strand breaks and double-strand repair activation. Taken together with our last publication, [7] we infer that the formation of adducts at dG, dC, and dA, along with a bulky hydroxy-benzethenoguanine adduct (Figure 1), is enough of a challenge to these cells that the lesion is difficult to repair and requires double-strand break repair during synthesis. This was validated by quantitative proteomics, which gave a strong indication of the mechanism, along with RNA-Seq results.…”

“…[5] We used an alternative and complementary approach based on selective activation by reactive oxygen species (ROS) to design next-generation smart DNA modifiers, termed ROS-activated cytotoxic agents (RACs). [6, 7] In this manuscript, we investigate the role and mechanism of DNA repair in treated cells to better understand the DNA modifying action of these novel RAC agents (Figure 1). …”

Arsenic trioxide amplifies cisplatin toxicity in human tubular cells transformed by HPV-16 E6/E7 for further therapeutic directions in renal cell carcinoma

“…By contrast, dysregulation of ATM network may promote oncogenesis by helping protect cancer cells (5), which may serve nefarious goals. As DNA damage is a major mechanism in chemotherapy, e.g., as represented by cisplatin (Cis-P) and other commonly used drugs, avoiding bystander toxicity to healthy cells via greater cancer specificity by chemically modified drugs has gathered interest (6). While insights into ATM-mediated DNA damage response (DDR) will help characterize tumor biology and provide therapeutic directions in RCC (7), these areas need more work.…”

Design of a hydrogen peroxide-activatable agent that specifically targets cancer cells

“…We hypothesized that simple anti-cancer agents that simply act through a conjugate addition would be more difficult to design due to in vivo metabolism and off-target effects thus we focused on a pro-drug strategy 8-10 . Certain cancer cells, for example acute myeloid leukemia (AML) cells, have elevated levels of ROS 11,12 .…”

“…A recent manuscript shows that induction of novel adducts changes anti-cancer activity and targets cancers 22 . The previous agents we designed possess mid-nanomolar activity against some tumor cells and up to 10-fold selectivity for AML cells 8,9 . Upon oxidation, the compound reacts by 1,2-addition with an amine identified as essential.…”