Self-Cyclizing Antioxidants to Prevent DNA Damage Caused by Hydroxyl Radical.

Antioxidant therapy is a promising treatment strategy for protecting DNA from the damage caused by reactive oxygen species (ROS). Here, we report new self-cyclizing antioxidant reagents that are selective for the hydroxyl radical. Our mechanistic investigation revealed that the reagents react with three equivalents of oxidant in a cascade reaction to form a bicyclic final product. Among the reagents synthesized, 1 c showed favorable properties in vitro and in cellular studies. Using As2 O3 , which triggers ROS production, we showed that 1 c prevents formation of the guanine oxidation product 2,2,4-triamino-2H-oxazol-5-one-2'-deoxyribonucleoside and lowers cellular levels of reactive oxygen. The described self-cyclizing antioxidants are efficient, flexible, and tunable reagents with the potential to limit toxic oxidative stress.

Excessive Reactive Oxygen Species and Exotic DNA Lesions as an Exploitable Liability.

Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the implications of oxidative stress are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with biomolecules. ROS cause activation of growth signals through thiol oxidation and may lead to DNA damage at elevated levels. In this review, we first discuss a conceptual framework for the interplay of ROS and antioxidants. This review then describes ROS signaling using FLT3-mediated growth signaling as an example. We then focus on ROS-mediated DNA damage. High concentrations of ROS result in various DNA lesions, including 8-oxo-7,8-dihydro-guanine, oxazolone, DNA-protein cross-links, and hydantoins, that have unique biological impacts. Here we delve into the biochemistry of nine well-characterized DNA lesions. Within each lesion, the types of repair mechanisms, the mutations induced, and their effects on transcription and replication are discussed. Finally, this review will discuss biochemically inspired implications for cancer therapy. Several teams have put forward designs to harness the excessive ROS and the burdened DNA repair systems of tumor cells for treating cancer. We discuss inhibition of the antioxidant system, the targeting of DNA repair, and ROS-activated prodrugs.

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

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8±0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells.

Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5'-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9±1µM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5'-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.

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

Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activate cell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactive oxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy we designed new hydrogen peroxide-activated agents with two key molecular pharmacophores. The first pharmacophore is a peroxide-acceptor and the second is a pendant amine. The acceptor is an N-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized that selectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesis and testing of fourteen compounds that differed at the pendent amine led to the identification of an agent (14) with 2µM activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ blood stem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water, with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminary mechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D and MLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed to activate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Western blotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selective ROS-activated anti-AML agent.

UV cell stress induces oxidative cyclization of a protective reagent for DNA damage reduction in skin explants.

UV irradiation is a major driver of DNA damage and ultimately skin cancer. UV exposure leads to persistent radicals that generate ROS over prolonged periods of time. Toward the goal of developing long-lasting antioxidants that can penetrate skin, we have designed a ROS-initiated protective (RIP) reagent that, upon reaction with ROS (antioxidant activity), self-cyclizes and then releases the natural product apocynin. Apocynin is a known antioxidant and inhibitor of NOX oxidase enzymes. A key phenol on the compound 1 controls ROS-initiated cyclization and makes 1 responsive to ROS with a EC50 comparable to common antioxidants in an ABTS assay. In an in vitro DNA nicking assay, the RIP reagent prevented DNA strand breaks. In cell-based assays, the reagent was not cytotoxic, apocynin was released only in cells treated with UVR, reduced UVR-induced cell death, and lowered DNA lesion formation. Finally, topical treatment of human skin explants with the RIP reagent reduced UV-induced DNA damage as monitored by quantification of cyclobutane dimer formation and DNA repair signaling via TP53. The reagent was more effective than administration of a catalase antioxidant on skin explants. This chemistry platform will expand the types of ROS-activated motifs and enable inhibitor release for potential use as a long-acting sunscreen.