A chemical-genetic screen identifies ABHD12 as an oxidized-phosphatidylserine lipase.

Reactive oxygen species (ROS) are transient, highly reactive intermediates or byproducts produced during oxygen metabolism. However, when innate mechanisms are unable to cope with sequestration of surplus ROS, oxidative stress results, in which excess ROS damage biomolecules. Oxidized phosphatidylserine (PS), a proapoptotic 'eat me' signal, is produced in response to elevated ROS, yet little is known regarding its chemical composition and metabolism. Here, we report a small molecule that generates ROS in different mammalian cells. We used this molecule to detect, characterize and study oxidized PS in mammalian cells. We developed a chemical-genetic screen to identify enzymes that regulate oxidized PS in mammalian cells and found that the lipase ABHD12 hydrolyzes oxidized PS. We validated these findings in different physiological settings including primary peritoneal macrophages and brains from Abhd12-/- mice under inflammatory stress, and in the process, we functionally annotated an enzyme regulating oxidized PS in vivo.

Targeted Antibacterial Activity Guided by Bacteria-Specific Nitroreductase Catalytic Activation to Produce Ciprofloxacin.

Fluoroquinolones (FQs) are among the front-line antibiotics used to treat severe infections caused by Gram-negative bacteria. However, recently, due to toxicity concerns, their use has been severely restricted. Hence, efforts to direct delivery of this antibiotic specifically to bacteria/site of infection are underway. Here, we report a strategy that uses a bacterial enzyme for activation of a prodrug to generate the active antibiotic. The ciprofloxacin-latent fluorophore conjugate 1, which is designed as a substrate for nitroreductase (NTR), a bacterial enzyme, was synthesized. Upon activation by NTR, release of Ciprofloxacin (CIP) as well as a fluorescence reporter was observed. We provide evidence for the prodrug permeating bacteria to generate a fluorescent signal and we found no evidence for activation in mammalian cells supporting selectivity of activation within bacteria. As a testament to its efficacy, 1 was found to have potent bactericidal activity nearly identical to CIP and significantly reduced the bacterial burden in a neutropenic mouse thigh infection model, again, at comparable potency with CIP, a clinically used FQ. Thus, together, we have developed a small molecule that facilitates bacteria-specific fluoroquinolone delivery.

FLUORO/NO: A Nitric Oxide Donor with a Fluorescence Reporter.

Nitric oxide (NO) plays significant signalling roles in cells; the controlled generation of NO is of therapeutic relevance. Although a number of methods for the delivery and detection of NO are available, these events are typically mutually exclusive. Furthermore, the efficiency of delivery of NO can be compromised by detection technologies that consume NO. Here, we report FLUORO/NO, an esterase-activated diazeniumdiolate-based NO donor with an in-built fluorescence reporter. We demonstrate that this compound is capable of enhancing NO within cells in a dose-dependent manner, accompanied by a similar increase in fluorescence. The compatibility of this tool to study NO-mediated signalling as well as NO-mediated stress is demonstrated. FLUORO/NO is a convenient tool that shows NO-like activity and allows monitoring of NO release. This tool will help interrogate the redox biology of NO.

Carbonyl Sulfide (COS) Donor Induced Protein Persulfidation Protects against Oxidative Stress.

The emergence of hydrogen sulfide (H2 S) as an important signalling molecule in redox biology with therapeutic potential has triggered interest in generating this molecule within cells. One strategy that has been proposed is to use carbonyl sulfide (COS) as a surrogate for hydrogen sulfide. Small molecules that generate COS have been shown to produce hydrogen sulfide in the presence of carbonic anhydrase, a widely prevalent enzyme. However, other studies have indicated that COS may have biological effects which are distinct from H2 S. Thus, it would be useful to develop tools to compare (and contrast) effects of COS and H2 S. Here we report enzyme-activated COS donors that are capable of inducing protein persulfidation, which is symptomatic of generation of hydrogen sulfide. The COS donors are also capable of mitigating stress induced by elevated reactive oxygen species. Together, our data suggests that the effects of COS parallel that of hydrogen sulfide, laying the foundation for further development of these donors as possible therapeutic agents.

Esterase Sensitive Self-Immolative Sulfur Dioxide Donors.

A series of cell-permeable esterase-sensitive sulfonates that undergo self-immolation to produce sulfur dioxide (SO2), a gaseous pollutant with new and emerging biological roles, is reported. These compounds should facilitate the study SO2 biology and will lay the platform for newer stimuli-responsive donors of this gas.

A small molecule for theraNOstic targeting of cancer cells.

Thera/NO - a small molecule that is activated by hydrogen peroxide to generate nitric oxide (NO) and a fluorescence signal is reported. Using cancer and primary cells, we show that Thera/NO preferentially releases NO in cancer cells, which can trigger DNA damage and cell death in them. The coupled fluorescence signal facilitated tracking the NO release in living cells without collateral consumption of NO.

Esterase Activated Carbonyl Sulfide/Hydrogen Sulfide (H2S) Donors.

Hydrogen sulfide (H2S) is a mediator of a number of cellular processes, and modulating cellular levels of this gas has emerged as an important therapeutic area. Localized generation of H2S is thus very useful but highly challenging. Here, we report pivaloyloxymethyl-based carbonothioates and carbamothioates that are activated by the enzyme, esterase, to generate carbonyl sulfide (COS), which is hydrolyzed to H2S.

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.