Synthesis and Evaluation of Small Molecule Based Reactive Oxygen Species (ROS) Generators

Abstract

Nearly all aerobic organisms use oxygen for respiration. Electrons released during respiration from redox enzymes are accidentally abstracted by O2 to produce superoxide (O2●–), which is subsequently converted to hydrogen peroxide (H2O2). In the presence of trace metal ions such as Fe2+ or Cu+, H2O2 can produce the highly reactive hydroxyl radical (OH●). Together, these species can react with the majority of biomacromolecules such as DNA, proteins and lipids, causing oxidative damage and are hence called reactive oxygen species (ROS). It is therefore not surprising that ROS are generated in immune cells to combat invading pathogens as a protective mechanism. Recently, ROS have been shown to sensitize infectious pathogens to clinical antibiotics suggesting that ROS-generating drugs could be used as antibacterials. With the rapid rise in bacteria acquiring drug resistance to most commercial antibiotics, development of new interventional strategies to help overcome drug resistance is critical. This however requires developing methodologies for the controlled generation of ROS. Although there are few methods available, none of them are suited for reliable ROS generation. Here, three distinct approaches for controlled ROS generation will be presented. First, a series of dihydrobenzoquinones were evaluated as sources of O2●–in physiological pH. These compounds are expected to undergo an enolization in buffer to produce a 1,4-dihydroxyarene, which are known to react with oxygen to generate ROS. In this series, ROS generation was found to depend in part on the propensity of the “keto” form of the dihydrobenzoquinone to enolize thus providing a structural handle to regulate rates of ROS generation. Having established the utility of these compounds to generate ROS in cell-free systems, we investigated the ability of these compounds to permeate cells to elevate ROS. Using an array of techniques, we provide evidence for these compounds to enhance ROS in bacteria including mycobacteria. We next exploited the utility of the ROS generator as an adjuvant to sensitize a Gram negative bacterium, E. coli to aminoglycoside antibiotics such as kanamycin and gentamycin. Next, a methodology for ROS generation triggered by a bacterial enzyme was developed. This enzyme activated ROS generator was found to permeate cells and enhance ROS levels inside bacteria. A spatiotemporally controlled generation of ROS was observed with this enzyme triggered ROS generator. Our comparative data suggests that this compound is better suited for the enhancement of intracellular ROS than the routinely used ROS sources. Finally, two scaffolds for thiol activated ROS generation were synthesized and studied. Here, an enhanced induction of oxidative stress is expected through depletion of intracellular antioxidant thiols and concomitant ROS generation. This synergestic effect was anticipated to be lethal to drug resistant bacteria. When tested, we found that a majority of the compounds were highly potent in vitro inhibitors of methicillin resistant Staphylococcus aureus supporting this as a viable strategy to overcome bacterial drug resistance.