The Glycolytic Metabolite Methylglyoxal Covalently Inactivates the NLRP3 Inflammasome.

The NLRP3 inflammasome promotes inflammation in disease, yet the full repertoire of mechanisms regulating its activity are not well delineated. Among established regulatory mechanisms, covalent modification of NLRP3 has emerged as a common route for pharmacological inactivation of this protein. Here, we show that inhibition of the glycolytic enzyme PGK1 results in the accumulation of methylglyoxal, a reactive metabolite whose increased levels decrease NLRP3 assembly and inflammatory signaling in cells. We find that methylglyoxal inactivates NLRP3 via a non-enzymatic, covalent crosslinking-based mechanism, promoting inter- and intra-protein MICA posttranslational linkages within NLRP3. This work establishes NLRP3 as capable of sensing a host of electrophilic chemicals, both exogenous small molecules and endogenous reactive metabolites, and suggests a mechanism by which glycolytic flux can moderate the activation status of a central inflammatory signaling pathway.

Enhancing cellular sulfane sulfur through ß-glycosidase-activated persulfide donors: mechanistic insights and oxidative stress mitigation.

Sulfane sulfur species such as persulfides and polysulfides along with hydrogen sulfide protect cells from oxidative stress and are key members of the cellular antioxidant pool. Here, we report perthiocarbamate-based prodrugs that are cleaved by ß-glycosidases to produce persulfide and relatively innocuous byproducts. The ß-glucosidase-activated persulfide donor enhances cellular sulfane sulfur and protects cells against lethality induced by elevated reactive oxygen species (ROS).

Leveraging an enzyme/artificial substrate system to enhance cellular persulfides and mitigate neuroinflammation.

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications.

A Vinyl-Boronate Ester-Based Persulfide Donor Controllable by Hydrogen Peroxide, a Reactive Oxygen Species (ROS).

A vinyl boronate ester-based persulfidating agent that is selectively activated by hydrogen peroxide, which is a reactive oxygen species (ROS), and efficiently generated a persulfide by a hitherto unexplored 1,4-O,S-relay mechanism is reported. This donor was found to protect cells from cytotoxicity induced by oxidants, and the major byproduct is cinnamaldehyde, which is widely used in the food industry as an additive.

Small molecule generators of biologically reactive sulfur species.

Sulfur metabolism is integral to cellular growth and survival. The presence of a wide range of oxidation states of sulfur in biology coupled with its unique reactivity are some key features of the biology of this element. In particular, nearly all oxidation states of sulfur not only occur but are also inter-convertible. In order to study the chemical biology of reactive sulfur species, tools to reliably detect as well as generate these species within cells are necessary. Herein, an overview of strategies to generate certain reactive sulfur species is presented. The donors of reactive sulfur species have been organized based on their oxidation states. These interesting small molecules have helped lay a strong foundation to study the biology of reactive sulfur species and some may have therapeutic applications in the future as well.

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.