ß-Galactosidase-activated nitroxyl (HNO) donors provide insights into redox cross-talk in senescent cells.

The cross-talk among reductive and oxidative species (redox cross-talk), especially those derived from sulfur, nitrogen and oxygen, influence several physiological processes including aging. One major hallmark of aging is cellular senescence, which is associated with chronic systemic inflammation. Here, we report a chemical tool that generates nitoxyl (HNO) upon activation by ß-galactosidase, an enzyme that is over-expressed in senescent cells. In a radiation-induced senescence model, the HNO donor suppressed reactive oxygen species (ROS) in a hydrogen sulfide (H2S)-dependent manner. Hence, the newly developed tool provides insights into redox cross-talk and establishes the foundation for new interventions that modulate levels of these species to mitigate oxidative stress and inflammation.

Reactive Sulfur Species in Biology and Medicine.

Hydrogen sulfide (H2S) has emerged as a third small-molecule bioactive signaling agent, along with nitric oxide (NO) and carbon monoxide (CO) [...].

Hydropersulfides (RSSH) attenuate doxorubicin-induced cardiotoxicity while boosting its anticancer action.

Cardiotoxicity is a frequent and often lethal complication of doxorubicin (DOX)-based chemotherapy. Here, we report that hydropersulfides (RSSH) are the most effective reactive sulfur species in conferring protection against DOX-induced toxicity in H9c2 cardiac cells. Mechanistically, RSSH supplementation alleviates the DOX-evoked surge in reactive oxygen species (ROS), activating nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways, thus boosting endogenous antioxidant defenses. Simultaneously, RSSH turns on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a master regulator of mitochondrial function, while decreasing caspase-3 activity to inhibit apoptosis. Of note, we find that RSSH potentiate anticancer DOX effects in three different cancer cell lines, with evidence that suggests this occurs via induction of reductive stress. Indeed, cancer cells already exhibit much higher basal hydrogen sulfide (H2S), sulfane sulfur, and reducing equivalents compared to cardiac cells. Thus, RSSH may represent a new promising avenue to fend off DOX-induced cardiotoxicity while boosting its anticancer effects.

Photochemical Release of Hydropersulfides.

Hydropersulfides (RSSH) have received significant interest in the field of redox biology because of their intriguing biochemical properties. However, because RSSH are inherently unstable, their study is challenging, and as a result, the details of their physiological roles remain ill-defined. Herein, we report strategies to release RSSH utilizing photoremovable protecting groups. RSSH protection with the well-established p-hydroxyphenacyl (pHP) photoprotecting group resulted in inefficient RSSH photorelease along with complex chemistry. Therefore, an alternative precursor was examined in which a self-immolative linker was inserted between the pHP group and RSSH, providing nearly quantitative RSSH release following photolysis at 365 nm. Inspired by these results, we also synthesized an analogous precursor derivatized with 7-diethylaminocoumarin (DEACM), a visible light-cleavable photoprotecting group. Photolysis of this precursor at 420 nm led to efficient RSSH release, and in vitro experiments demonstrated intracellular RSSH delivery in breast cancer MCF-7 cells.

Hydropersulfides Inhibit Lipid Peroxidation and Protect Cells from Ferroptosis.

Hydropersulfides (RSSH) are believed to serve important roles in vivo, including as scavengers of damaging oxidants and electrophiles. The α-effect makes RSSH not only much better nucleophiles than thiols (RSH), but also much more potent H-atom transfer agents. Since HAT is the mechanism of action of the most potent small-molecule inhibitors of phospholipid peroxidation and associated ferroptotic cell death, we have investigated their reactivity in this context. Using the fluorescence-enabled inhibited autoxidation (FENIX) approach, we have found RSSH to be highly reactive toward phospholipid-derived peroxyl radicals (kinh = 2 × 105 M-1 s-1), equaling the most potent ferroptosis inhibitors identified to date. Related (poly)sulfide products resulting from the rapid self-reaction of RSSH under physiological conditions (e.g., disulfide, trisulfide, H2S) are essentially unreactive, but combinations from which RSSH can be produced in situ (i.e., polysulfides with H2S or thiols with H2S2) are effective. In situ generation of RSSH from designed precursors which release RSSH via intramolecular substitution or hydrolysis improve the radical-trapping efficiency of RSSH by minimizing deleterious self-reactions. A brief survey of structure-reactivity relationships enabled the design of new precursors that are more efficient. The reactivity of RSSH and their precursors translates from (phospho)lipid bilayers to cell culture (mouse embryonic fibroblasts), where they were found to inhibit ferroptosis induced by inactivation of glutathione peroxidase-4 (GPX4) or deletion of the gene encoding it. These results suggest that RSSH and the pathways responsible for their biosynthesis may act as a ferroptosis suppression system alongside the recently discovered FSP1/ubiquinone and GCH1/BH4/DHFR systems.

The reaction of hydropersulfides (RSSH) with S-nitrosothiols (RS-NO) and the biological/physiological implications.

S-Nitrosothiol (RS-NO) generation/levels have been implicated as being important to numerous physiological and pathophysiological processes. As such, the mechanism(s) of their generation and degradation are important factors in determining their biological activity. Along with the effects on the activity of thiol proteins, RS-NOs have also been reported to be reservoirs or storage forms of nitric oxide (NO). That is, it is hypothesized that NO can be released from RS-NO at opportune times to, for example, regulate vascular tone. However, to date there are few established mechanisms that can account for facile NO release from RS-NO. Recent discovery of the biological formation and prevalence of hydropersulfides (RSSH) and their subsequent reaction with RS-NO species provides a possible route for NO release from RS-NO. Herein, it is found that RSSH is capable of reacting with RS-NO to liberate NO and that the analogous reaction using RSH is not nearly as proficient in generating NO. Moreover, computational results support the prevalence of this reaction over other possible competing processes. Finally, results of biological studies of NO-mediated vasorelaxation are consistent with the idea that RS-NO species can be degraded by RSSH to release NO.

Hydropersulfides (RSSH) Outperform Post-Conditioning and Other Reactive Sulfur Species in Limiting Ischemia-Reperfusion Injury in the Isolated Mouse Heart.

Hydrogen sulfide (H2S) exhibits protective effects in cardiovascular disease such as myocardial ischemia/reperfusion (I/R) injury, cardiac hypertrophy, and atherosclerosis. Despite these findings, its mechanism of action remains elusive. Recent studies suggest that H2S can modulate protein activity through redox-based post-translational modifications of protein cysteine residues forming hydropersulfides (RSSH). Furthermore, emerging evidence indicates that reactive sulfur species, including RSSH and polysulfides, exhibit cardioprotective action. However, it is not clear yet whether there are any pharmacological differences in the use of H2S vs. RSSH and/or polysulfides. This study aims to examine the differing cardioprotective effects of distinct reactive sulfur species (RSS) such as H2S, RSSH, and dialkyl trisulfides (RSSSR) compared with canonical ischemic post-conditioning in the context of a Langendorff ex-vivo myocardial I/R injury model. For the first time, a side-by-side study has revealed that exogenous RSSH donation is a superior approach to maintain post-ischemic function and limit infarct size when compared with other RSS and mechanical post-conditioning. Our results also suggest that RSSH preserves mitochondrial respiration in H9c2 cardiomyocytes exposed to hypoxia-reoxygenation via inhibition of oxidative phosphorylation while preserving cell viability.

Hydropersulfides (RSSH) and Nitric Oxide (NO) Signaling: Possible Effects on S-Nitrosothiols (RS-NO).

S-Nitrosothiol (RS-NO) formation in proteins and peptides have been implicated as factors in the etiology of many diseases and as possible regulators of thiol protein function. They have also been proposed as possible storage forms of nitric oxide (NO). However, despite their proposed functions/roles, there appears to be little consensus regarding the physiological mechanisms of RS-NO formation and degradation. Hydropersulfides (RSSH) have recently been discovered as endogenously generated species with unique reactivity. One important reaction of RSSH is with RS-NO, which leads to the degradation of RS-NO as well as the release of NO. Thus, it can be speculated that RSSH can be a factor in the regulation of steady-state RS-NO levels, and therefore may be important in RS-NO (patho)physiology. Moreover, RSSH-mediated NO release from RS-NO may be a possible mechanism allowing RS-NO to serve as a storage form of NO.

Development of Hydropersulfide Donors to Study Their Chemical Biology.

Significance: Hydropersulfides (RSSH) are ubiquitous in prokaryotes, eukaryotic cells, and mammalian tissues. The unique chemical properties and prevalent nature of these species suggest a crucial role of RSSH in cell regulatory processes, yet little is known about their physiological functions. Recent Advances: Examining the biological roles of RSSH species is challenging because of their inherent instability. In recent years, researchers have developed a number of small-molecule donors that efficiently release RSSH in response to various stimuli, including pH, thiols, reactive oxygen species, enzymes, and light. These RSSH donors have provided researchers with chemical tools to uncover the potential function and role of RSSH as physiological signaling and/or protecting agents. Critical Issues: Because RSSH, hydrogen sulfide (H2S), and higher order polysulfides are related to each other and can be present simultaneously in biological systems, distinguishing among the activities due to each of these species is difficult. Discerning this activity is critical to elucidate the chemical biology and physiology of RSSH. Moreover, although RSSH donors have been shown to confer cytoprotection against oxidative and electrophilic stress, their biological targets remain to be elucidated. Future Directions: The development of RSSH donors with optimal drug-like properties and selectivity toward specific tissues/pathologies represents a promising approach. Further investigation of releasing efficiencies in vivo and a clear understanding of RSSH biological responses remain targets for future investigation. Antioxid. Redox Signal. 36, 309-326.

Solid-gas reactions for nitroxyl (HNO) generation in the gas phase.

We present a novel nitroxyl (HNO) generation method, which avoids the need of using a liquid system or extreme experimental conditions. This method consists of the reaction between a gaseous base and an HNO donor (Piloty's acid) in the solid phase, allowing the formation of gaseous HNO in a fast and economical way. Detection of HNO was carried out indirectly, measuring the nitrous oxide (N2O) byproduct of HNO dimerization using infrared spectroscopy, and directly, using mass spectrometry techniques and an electrochemical HNO sensor.

Alkylsulfenyl thiocarbonates: precursors to hydropersulfides potently attenuate oxidative stress.

The recent discovery of the prevalence of hydropersulfides (RSSH) species in biological systems suggests their potential roles in cell regulatory processes. However, the reactive and transient nature of RSSH makes their study difficult, and dependent on the use of donor molecules. Herein, we report alkylsulfenyl thiocarbonates as a new class of RSSH precursors that efficiently release RSSH under physiologically relevant conditions. RSSH release kinetics from these precursors are tunable through electronic modification of the thiocarbonate carbonyl group's electrophilicity. In addition, these precursors also react with thiols to release RSSH with a minor amount of carbonyl sulfide (COS). Importantly, RSSH generation by these precursors protects against oxidative stress in H9c2 cardiac myoblasts. Furthermore, we demonstrate the ability of these precursors to increase intracellular RSSH levels.

Reaction of Nitroxyl (HNO) with Hydrogen Sulfide and Hydropersulfides.

Nitroxyl (HNO) has gained a considerable amount of attention because of its promising pharmacological effects. The biochemical mechanisms of HNO activity are associated with the modification of regulatory thiol proteins. Recently, several studies have suggested that hydropersulfides (RSSH), presumed signaling products of hydrogen sulfide (H2S)-mediated thiol (RSH) modification, are additional potential targets of HNO. However, the interaction of HNO with reactive sulfur species beyond thiols remains relatively unexplored. Herein, we present characterization of HNO reactivity with H2S and RSSH. The reaction of H2S with HNO leads to the formation of hydrogen polysulfides and sulfur (S8), suggesting a potential role in sulfane sulfur homeostasis. Furthermore, we show that hydropersulfides are more efficient traps for HNO than their thiol counterparts. The reaction of HNO with RSSH at varied stoichiometries has been examined with the observed production of various dialkylpolysulfides (RSSnSR) and other nitrogen-containing dialkylpolysulfide species (RSS-NH-SnR). We do not observe evidence of sulfenylsulfinamide (RS-S(O)-NH2) formation, a pathway expected by analogy with the known reactivity of HNO with thiol.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

Predicting the Possible Physiological/Biological Utility of the Hydropersulfide Functional Group Based on Its Chemistry: Similarities Between Hydropersulfides and Selenols.

Significance: Hydropersulfides (RSSH) and related polysulfide species (RSnR, n > 2, R = alkyl, H) are highly biologically prevalent with likely important physiological functions. Due to their prevalence, many labs have begun to investigate their possible roles, especially with regards to their protective, redox, and signaling properties. Recent Advances: A significant amount of work has been performed while delineating the chemical reactivity/chemical properties of hydropersulfides, and it is clear that their overall chemistry is distinct from all other biologically relevant sulfur species (e.g., thiols, disulfides, sulfenic acids, etc.). Critical Issues: One way to predict and ultimately understand the biological functions of hydropersulfides is to focus on their unique chemistry, which should provide the rationale for why this unique functionality is present. Interestingly, some of the chemical properties of RSSH are strikingly similar to those of selenols (RSeH). Therefore, it may be important to consider the known functions of selenoproteins when speculating about the possible functions of RSSH species. Future Directions: Currently, many of the inherent chemical differences between hydropersulfides and other biological sulfur species have been established. It remains to be determined, however, whether and how these differences are utilized to accomplish specific biochemical/physiological goals. A significant aspect of elucidating the biological utility of hydropersulfides will be to determine the mechanisms of regulation of their formation and/or biosynthesis, that is, based on whether it can be determined under what cellular conditions hydropersulfides are made, more meaningful speculation regarding their functions/roles can be developed.

Alkylamine-Substituted Perthiocarbamates: Dual Precursors to Hydropersulfide and Carbonyl Sulfide with Cardioprotective Actions.

The recent discovery of hydropersulfides (RSSH) in mammalian systems suggests their potential roles in cell signaling. However, the exploration of RSSH biological significance is challenging due to their instability under physiological conditions. Herein, we report the preparation, RSSH-releasing properties, and cytoprotective nature of alkylamine-substituted perthiocarbamates. Triggered by a base-sensitive, self-immolative moiety, these precursors show efficient RSSH release and also demonstrate the ability to generate carbonyl sulfide (COS) in the presence of thiols. Using this dually reactive alkylamine-substituted perthiocarbamate platform, the generation of both RSSH and COS is tunable with respect to half-life, pH, and availability of thiols. Importantly, these precursors exhibit cytoprotective effects against hydrogen peroxide-mediated toxicity in H9c2 cells and cardioprotective effects against myocardial ischemic/reperfusion injury, indicating their potential application as new RSSH- and/or COS-releasing therapeutics.

Nitroxyl (HNO) targets phospholamban cysteines 41 and 46 to enhance cardiac function.

Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

The reaction of hydrogen sulfide with disulfides: formation of a stable trisulfide and implications for biological systems.

BACKGROUND AND PURPOSE: The signalling associated with hydrogen sulfide (H2 S) remains to be established, and recent studies have alluded to the possibility that H2 S-derived species play important roles. Of particular interest are hydropersulfides (RSSH) and related polysulfides (RSSn R, n > 1). This work elucidates the fundamental chemical relationship between these sulfur species as well as examines their biological effects. EXPERIMENTAL APPROACH: Using standard analytical techniques (1 H-NMR and MS), the equilibrium reactions between H2 S, disulfides (RSSR), RSSH, dialkyltrisulfides (RSSSR) and thiols (RSH) were examined. Their ability to protect cells from electrophilic and/or oxidative stress was also examined using cell culture. KEY RESULTS: H2 S, RSSR, RSSH, RSSSR and RSH are all in a dynamic equilibrium. In a biological system, these species can exist simultaneously, and thus, it is difficult to discern which species is (are) the biological effector(s). Treatment of cells with the dialkyl trisulfide cysteine trisulfide (Cys-SSS-Cys) resulted in high intracellular levels of hydropersulfides and protection from electrophilic stress. CONCLUSIONS AND IMPLICATIONS: In aqueous systems, the reaction between H2 S and RSSR results in the formation of equilibria whereby H2 S, RSH, RSSR, RSSH and RSSSR are present. In a biological system, any of these species can be responsible for the observed biological activity. These equilibrium species can also be generated via the reaction of RSH with RSSSR. Due to these equilibria, Cys-SSS-Cys can be a method for generating any of the other species. Importantly, HEK293T cells treated with Cys-SSS-Cys results in increased levels of hydropersulfides, allowing examination of the biological effects of RSSH. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Development of S-Substituted Thioisothioureas as Efficient Hydropersulfide Precursors.

Because of their inherent instability, hydropersulfides (RSSH) must be generated in situ using precursors, but very few physiologically useful RSSH precursors have been developed to date. In this work, we report the design, synthesis, and evaluation of novel S-substituted thiosiothioureas as RSSH precursors. These water-soluble precursors show efficient and controllable release of RSSH under physiological conditions.

Chemical Biology of Hydropersulfides and Related Species: Possible Roles in Cellular Protection and Redox Signaling.

SIGNIFICANCE: For >20 years, physiological signaling associated with the endogenous generation of hydrogen sulfide (H2S) has been of significant interest. Despite its presumed importance, the biochemical mechanisms associated with its actions have not been elucidated. Recent Advances: Recently it has been found that H2S-related or derived species are highly prevalent in mammalian systems and that these species may be responsible for some, if not the majority, of the biological actions attributed to H2S. One of the most prevalent and intriguing species are hydropersulfides (RSSH), which can be present at significant levels. Indeed, it appears that H2S and RSSH are intimately linked in biological systems and likely to be mutually inclusive. CRITICAL ISSUES: The fact that H2S and polysulfides such as RSSH are present simultaneously means that the biological actions previously assigned to H2S can be instead because of the presence of RSSH (or other polysulfides). Thus, it remains possible that hydropersulfides are the biological effectors, and H2S serves, to a certain extent, as a marker for persulfides and polysulfides. Addressing this possibility will to a large extent be based on the chemistry of these species. FUTURE DIRECTIONS: Currently, it is known that persulfides possess unique and novel chemical properties that may explain their biological prevalence. However, significantly more work will be required to establish the possible physiological roles of these species. Moreover, an understanding of the regulation of their biosynthesis and degradation will become important topics in piecing together their biology. Antioxid. Redox Signal. 00, 000-000.