A DFT examination of the role of proximal boron functionalities in the S-alkylation of sulfenic acid anions

DFT modelling predicts proximal boron groups can accelerate sulfenate alkylation reactions, depending on boron substituents and boron distance from the reaction site.

Reaction-based fluorogenic probes for selective detection of cysteinyl oxidation in living cells

Measuring reactive oxygen, nitrogen and sulfur species in cells is established technology, but turn-on fluorescence tools for detecting the products of their reaction with protein cysteines remain essentially unknown. Toward this goal, here we describe fluorogenic probes for sulfenic acid, a redox modification of protein cysteines inextricably linked to signaling and oxidative stress. The probes, called CysOx1 and CysOx2, are reaction-based, exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We applied CysOx2 in a cell-based 96-well plate assay to determine whether kinase inhibitors modulate protein S-sulfenylation as well as O-phosphorylation. Analysis of these data revealed an unexpected positive association of S-sulfenylation and inhibition of select kinases within the TK, AGC, and CMGC families including GSK3, a multitasking Ser/Thr kinase and emerging therapeutic target for neurodegenerative and mood disorders. Chemoproteomic mapping of sulfenic acid-modified cysteines in GSK3 inhibitor-treated cells shows that sites of S-oxidation localize to regulatory cysteines within key components of antioxidant defense systems. Our studies with CysOx probes offer up new insights into kinase-inhibitor dependent modulation of sulfenylome dynamics and should accelerate future efforts in the modern era of translational redox medicine.

H2O2-Mediated Oxidative Stress Enhances Cystathionine γ-Lyase-Derived H2S Synthesis via a Sulfenic Acid Intermediate

Hydrogen sulfide (H2S), which is generated mainly by cystathionine γ-lyase (CSE) in the cardiovascular system, plays a pivotal role in a wide range of physiological and pathological processes. However, the regulatory mechanism of the CSE/H2S system is poorly understood. Herein, we show that oxidation induces the disulfide bond formation between Cys252 and Cys255 in the CXXC motif, thus stimulating the H2S-producing activity of CSE. The activity of oxidized CSE is approximately 2.5 fold greater than that of the reduced enzyme. Molecular dynamics and molecular docking suggest that the disulfide bond formation induces the conformational change in the active site of CSE and consequently increases the affinity of the enzyme for the substrate L-cysteine. Mass spectrometry and mutagenesis studies further established that the residue Cys255 is crucial for oxidation sensing. Oxidative stress-mediated sulfenylation of Cys255 leads to a sulfenic acid intermediate that spontaneously forms an intramolecular disulfide bond with the vicinal thiol group of Cys252. Moreover, we demonstrate that exogenous hydrogen peroxide (H2O2) and endogenous H2O2 triggered by vascular endothelial growth factor (VEGF) promote cellular H2S production through the enhancement of CSE activity under oxidative stress conditions. By contrast, incubation with H2O2 or VEGF did not significantly enhance cellular H2S production in the presence of PEG-catalase, an enzymatic cell-permeable H2O2 scavenger with high H2O2 specificity. Taken together, we report a new posttranslational modification of CSE that provides a molecular mechanism for H2O2/H2S crosstalk in cells under oxidative stress.

Kinetic Studies of Antioxidant Properties of Ovothiol A

Ovothiol A (OSH) is one of the strongest natural antioxidants. So far, its presence was found in tissues of marine invertebrates, algae and fish. Due to very low pKa value of the SH group, under physiological conditions, this compound is almost entirely present in chemically active thiolate form and reacts with ROS and radicals significantly faster than other natural thiols. In biological systems, OSH acts in tandem with glutathione GSH, with OSH neutralizing oxidants and GSH maintaining ovothiol in the reduced state. In the present work, we report the rate constants of OSH oxidation by H2O2 and of reduction of oxidized ovothiol OSSO by GSH and we estimate the Arrhenius parameters for these rate constants. The absorption spectra of reaction intermediates, adduct OSSG and sulfenic acid OSOH, were obtained. We also found that OSH effectively quenches the triplet state of kynurenic acid with an almost diffusion-controlled rate constant. This finding indicates that OSH may serve as a good photoprotector to inhibit the deleterious effect of solar UV irradiation; this assumption explains the high concentrations of OSH in the fish lens. The unique antioxidant and photoprotecting properties of OSH open promising perspectives for its use in the treatment of human diseases.

Polymorphisms of Genes Related to Phase-II Metabolism and the Resistance of Clopidogrel

Clopidogrel is one of the thienopyridine antiplatelet drugs commonly used as a prophylactic medication to prevent coagulation in vessels and cardiovascular events. The molecule of clopidogrel is metabolized in the liver via phase-I and phase-II metabolism pathways. The sulfenic acid clopidogrel metabolite undergoes phase-II metabolism through conjugation with glutathione by the glutathione-s-transferase (GST) to form a glutathione conjugate of clopidogrel (inactive metabolite). A glutaredoxin enzyme removes the glutathione conjugated with clopidogrel to form cis-thiol-clopidogrel. This review focused on the polymorphisms of genes related to phase-II metabolism during the clopidogrel bioactivation process. Overall, no well-controlled studies were done about the relationship between the clopidogrel bioactivation process and genes related to phase-II metabolism’s enzymes. Nevertheless, some polymorphisms of G6PD, GCLC, GCLM, GSS, GST, GSR, HK, and GLRX genes could be responsible for clopidogrel resistance due to low glutathione conjugate or glutaredoxin plasma levels. Studies needed to be concerned with the relationship between clopidogrel resistance and phase-II metabolism issues in the near future.

Parallel Evaluation of Nucleophilic and Electrophilic Chemical Probes for Sulfenic Acid: Reactivity, Selectivity and Biocompatibility

S-sulfenylation of cysteine thiols (Cys-SOH) is a regulatory posttranslational modification in redox signaling and an important intermediate to other cysteine chemotypes. Owing to the dual chemical nature of the sulfur in sulfenic acid, both nucleophilic and electrophilic chemical probes have been developed to react with and detect Cys-SOH; however, the efficiency of existing probes has not been evaluated in a side-by-side comparison. Here, we employ small-molecule and protein models of Cys-SOH and compare the chemical probe reactivity. These data clearly show that 1,3-diketone-based nucleophilic probes react more efficiently with sulfenic acid as compared to strained alkene/alkyne electrophilic probes. Kinetic experiments that rigorously address the selectivity of the 1,3-diketone-based probes are also reported. Consideration of these data alongside relative cellular abundance, indicates that biological electrophiles, including cyclic sulfenamides, aldehydes, disulfides and hydrogen peroxide, are not meaningful targets of 1,3-diketone-based nucleophilic probes, which still remain the most viable tools for the bioorthogonal detection of Cys-SOH.

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.