Chemoproteomics of an Indole-Based Quinone Epoxide Identifies Druggable Vulnerabilities in Vancomycin-Resistant Staphylococcus aureus.

The alarming global rise in fatalities from multidrug-resistant Staphylococcus aureus (S. aureus) infections has underscored a need to develop new therapies to address this epidemic. Chemoproteomics is valuable in identifying targets for new drugs in different human diseases including bacterial infections. Targeting functional cysteines is particularly attractive, as they serve critical catalytic functions that enable bacterial survival. Here, we report an indole-based quinone epoxide scaffold with a unique boat-like conformation that allows steric control in modulating thiol reactivity. We extensively characterize a lead compound (4a), which potently inhibits clinically derived vancomycin-resistant S. aureus. Leveraging diverse chemoproteomic platforms, we identify and biochemically validate important transcriptional factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus, the chemical tools and biological targets that we describe here prospect new therapeutic paradigms in combatting S. aureus infections.

Role of Reactive Oxygen Species (ROS) in Therapeutics and Drug Resistance in Cancer and Bacteria.

Evading persistent drug resistance in cancer and bacteria is quintessential to restore health in humans, and impels intervention strategies. A distinct property of the cancer phenotype is enhanced glucose metabolism and oxidative stress. Reactive oxygen species (ROS) are metabolic byproducts of aerobic respiration and are responsible for maintaining redox homeostasis in cells. Redox balance and oxidative stress are orchestrated by antioxidant enzymes, reduced thiols and NADP(H) cofactors, which is critical for cancer cells survival and progression. Similarly, Escherichia coli (E. coli) and life-threatening infectious pathogens such as Staphylococcus aureus (SA) and Mycobacterium tuberculosis (Mtb) are appreciably sensitive to changes in the intracellular oxidative environment. Thus, small molecules that modulate antioxidant levels and/or enhance intracellular ROS could disturb the cellular oxidative environment and induce cell death, and hence could serve as novel therapeutics. Presented here are a collection of approaches that involve ROS modulation in cells as a strategy to target cancer and bacteria.

Mycobacterium tuberculosis has diminished capacity to counteract redox stress induced by elevated levels of endogenous superoxide.

Mycobacterium tuberculosis (Mtb) has evolved protective and detoxification mechanisms to maintain cytoplasmic redox balance in response to exogenous oxidative stress encountered inside host phagocytes. In contrast, little is known about the dynamic response of this pathogen to endogenous oxidative stress generated within Mtb. Using a noninvasive and specific biosensor of cytoplasmic redox state of Mtb, we for first time discovered a surprisingly high sensitivity of this pathogen to perturbation in redox homeostasis induced by elevated endogenous reactive oxygen species (ROS). We synthesized a series of hydroquinone-based small molecule ROS generators and found that ATD-3169 permeated mycobacteria to reliably enhance endogenous ROS including superoxide radicals. When Mtb strains including multidrug-resistant (MDR) and extensively drug-resistant (XDR) patient isolates were exposed to this compound, a dose-dependent, long-lasting, and irreversible oxidative shift in intramycobacterial redox potential was detected. Dynamic redox potential measurements revealed that Mtb had diminished capacity to restore cytoplasmic redox balance in comparison with Mycobacterium smegmatis (Msm), a fast growing nonpathogenic mycobacterial species. Accordingly, Mtb strains were extremely susceptible to inhibition by ATD-3169 but not Msm, suggesting a functional linkage between dynamic redox changes and survival. Microarray analysis showed major realignment of pathways involved in redox homeostasis, central metabolism, DNA repair, and cell wall lipid biosynthesis in response to ATD-3169, all consistent with enhanced endogenous ROS contributing to lethality induced by this compound. This work provides empirical evidence that the cytoplasmic redox poise of Mtb is uniquely sensitive to manipulation in steady-state endogenous ROS levels, thus revealing the importance of targeting intramycobacterial redox metabolism for controlling TB infection.

A phenacrylate scaffold for tunable thiol activation and release.

A thiol-selective 2-methyl-3-phenacrylate scaffold with spatiotemporal control over delivery of a cargo is reported. The half-lives of decomposition could be tuned from 30 min to 1 day and the scaffold's utility in thiol-inducible fluorophore release in cell-free as well as within cells is demonstrated.

Substituent effects on reactive oxygen species (ROS) generation by hydroquinones.

In order to understand the structural aspects of stabilization of hydroquinones and their ability to generate reactive oxygen species (ROS), we designed and synthesized a series of 6-aryl-2,3-dihydro-1,4-benzoquinones. These compounds equilibrate with the corresponding 6-aryl-1,4-dihydroxybenzenes in an organic medium; a linear free energy relationship analysis gave ρ = +2.37, suggesting that this equilibrium was sensitive to electronic effects. The propensity of the compound to enolize appears to determine ROS-generating capability, thus offering scope for tunable ROS generation.

Arylboronate ester based diazeniumdiolates (BORO/NO), a class of hydrogen peroxide inducible nitric oxide (NO) donors.

Here, we report the design, synthesis, and evaluation of arylboronate ester based diazeniumdiolates (BORO/NO), a class of nitric oxide (NO) donors activated by hydrogen peroxide (H2O2), a reactive oxygen species (ROS), to generate NO. We provide evidence for the NO donors' ability to permeate bacteria to produce NO when exposed to H2O2 supporting possible applications for BORO/NO to study molecular mechanisms of NO generation in response to elevated ROS.

A small molecule for controlled generation of reactive oxygen species (ROS).

Due to the short half-life of reactive oxygen species (ROS) such as a superoxide radical, controlled and localized generation of ROS is challenging. Here, we report a rationally designed small-molecule 1c that generates ROS only when triggered by a bacterial enzyme. We provide evidence for 1c predictably enhancing the intracellular superoxide radical in a model bacterium. Spatiotemporal control over ROS generation offered by 1c should help better understand stress responses in bacteria to increased ROS.

Design, synthesis and evaluation of small molecule reactive oxygen species generators as selective Mycobacterium tuberculosis inhibitors.

Here, we report 5-hydroxy-1,2,3,4,4a,9a-hexahydro-1,4-ethano-9,10-anthraquinone (13), a small molecule generating reactive oxygen species (ROS) in pH 7.4 buffer under ambient aerobic conditions that has selective and potent Mycobacterium tuberculosis growth inhibitory activity.

Synthesis, reactive oxygen species generation and copper-mediated nuclease activity profiles of 2-aryl-3-amino-1,4-naphthoquinones.

Here we report a series of 2-aryl-3-amino-1,4-naphthoquinones that generated reactive oxygen species (ROS) such as superoxide and hydrogen peroxide upon incubation in pH 7.4 under ambient aerobic conditions. ROS generation from these compounds was sensitive to structural modifications at the 3-amino position and a 2-aryl substituent promoted ROS generation. A number of these compounds were found to induce DNA damage in the presence of Cu(II) without any added reducing agent. Our data suggests that 2-aryl-3-amino-1,4-naphthoquinones' propensity to produce ROS correlated well with its DNA damage inducing ability. 2-Phenyl-3-pyrrolid-1-yl-1,4-naphthoquinone (22) was found to damage DNA at 1 µM suggesting that these compounds may have therapeutic relevance in targeting cancers which over-express Cu(II).