Synthesis and Evaluation of Small Molecule Generators of Redox-Active Reactive Species

Abstract

Chemically reactive species derived from oxygen, nitrogen and sulfur are produced in nearly all living cells and play important roles in numerous physiological processes. Recently, several studies have demonstrated that reactive oxygen species (ROS), reactive nitrogen species (RNS) which includes peroxynitrite (ONOO-) and hydrogen sulfide (H2S) are involved in host-pathogen interaction and antibiotic action. However, the precise role of these redox-active reactive species in bacteria remains poorly characterized. This is, in part, due to transient nature of these reactive species. In order to study the precise role of these reactive species in bacteria, their reliable sources would be useful. Although numerous methodologies for generating ROS, ONOO- and H2S have been reported, they are typically associated with various limitations such as poor cell permeability and lack of specificity. Here, we propose to design and develop small molecules that can reliably generate each of these reactive species independently within cells and study their roles in bacterial growth inhibition and antibiotic resistance. First, in order to generate ROS within bacteria, a series of natural product-inspired benzo[b]phenanthridine-5,7,12-triones were synthesized and studied. These compounds were found to generate ROS only in the presence of a bioreductive enzyme and were capable of enhancing ROS in Staphylococcus aureus (S. aureus). Several ROS generators from this series showed moderate to potent inhibitory activity against drug-resistant strain of S. aureus suggesting that antibiotic resistance in certain bacteria can be targeted by perturbing bacterial redox-homeostasis.

Next, we designed and synthesized a bioreductively activated ONOO- generator, in which a superoxide generating 1,4-naphthoquinone scaffold is strategically linked to a diazeniumdiolate-based nitric oxide donor. Using an array of experiments, the ability of this small molecule to generate ONOO- upon reaction with a bioreductive enzyme was demonstrated. The superiority of this compound to enhance ONOO- levels in both cell-free as well as within cells was demonstrated. Our results support the utility of this compound to study the role of ONOO- in bacteria.

Lastly, in order to selectively generate H2S within bacteria, bis(4-nitrobenzyl)sulfanes were designed and synthesized as nitroreductase (NTR) activated H2S donors. These compounds were found to be metabolized by NTR to produce H2S. The suitability of these compounds to enhance H2S levels in both Gram-positive as well as Gram-negative bacteria was demonstrated. The tools developed here are well suited to interrogate the role of H2S in antibiotic resistance.

In summary, our results address important problems associated with site-specific delivery of ROS, ONOO- and H2S within cells and we anticipate that these small molecules can be used as tools to understand the precise roles of these reactive species in antibiotic resistance.