Control of Oxidative Posttranslational Cysteine Modifications: From

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Control of Oxidative Posttranslational Cysteine Modifications: From Intricate Chemistry to Widespread Biological and Medical Applications Claus Jacob,*,† Eric Battaglia,‡ Torsten Burkholz,† Du Peng,† Denyse Bagrel,‡ and Mathias Montenarh§ †

Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, D-66123 Saarbruecken, Germany Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Université Paul Verlaine-Metz, 57070 Metz, France § Medical Biochemistry and Molecular Biology, Saarland University, Building 44, D-66424 Homburg, Germany ‡

ABSTRACT: Cysteine residues in proteins and enzymes often fulfill rather important roles, particularly in the context of cellular signaling, protein−protein interactions, substrate and metal binding, and catalysis. At the same time, some of the most active cysteine residues are also quite sensitive toward (oxidative) modification. S-Thiolation, S-nitrosation, and disulfide bond and sulfenic acid formation are processes which occur frequently inside the cell and regulate the function and activity of many proteins and enzymes. During oxidative stress, such modifications trigger, among others, antioxidant responses and cell death. The unique combination of nonredox function on the one hand and participation in redox signaling and control on the other has placed many cysteine proteins at the center of drug design and pesticide development. Research during the past decade has identified a range of chemically rather interesting, biologically very active substances that are able to modify cysteine residues in such proteins with huge efficiency, yet also considerable selectivity. These agents are often based on natural products and range from simple disulfides to complex polysulfanes, tetrahydrothienopyridines, α,β -unsaturated disulfides, thiuramdisulfides, and 1,2-dithiole-3-thiones. At the same time, inhibition of enzymes responsible for posttranslational cysteine modifications (and their removal) has become an important area of innovative drug research. Such investigations into the control of the cellular thiolstat by thiol-selective agents cross many disciplines and are often far from trivial.

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CONTENTS

1. Introduction 2. Interfering with Cysteine Proteins 3. From Simple Disulfides to Highly Reactive Sulfur Species 4. Techniques to Probe the Reactivity of Cysteine and Its Posttranslational Modifications 5. Aspects of the Biochemical Impact of Posttranslational Cysteine Modifications 6. Conclusions Author Information Corresponding Author Funding Acknowledgments Abbreviations References © 2011 American Chemical Society

1. INTRODUCTION The past decade has witnessed the emergence of a range of compounds which are able to modify cysteine residues in proteins and enzymes with considerable selectivity. Some of these cysteine-modifying agents bear considerable promise in the field of drug design and pesticide development. Substances such as the polysulfanes, prazoles, anethole dithiolethione (Sulfarlem), 4-methyl-5-(2-pyrazinyl)-3-dithiolethione (Oltipraz), and (+)-(S)-methyl-2-(2-chlorophenyl)-2-(6,7dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (Clopidogrel), for instance, are already in medical use for the treatment of various human disorders, while other compounds are in the pipeline (Figure 1). The emergence of these agents is closely

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Received: August 12, 2011 Published: November 22, 2011 588

dx.doi.org/10.1021/tx200342b | Chem. Res. Toxicol. 2012, 25, 588−604

Chemical Research in Toxicology

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Figure 1. Thiol/disulfide reactions involving reactive disulfides provide considerable selectivity for cysteine thiols in numerous proteins and enzymes. Since disulfides often are not reactive enough, research has developed a range of structures which are derived from the initial disulfide motif yet are considerably more reactive. This scheme highlights some of these unusual sulfur species. Where appropriate, it showcases examples of already existing drugs or agents currently under investigation. Please note that some of these agents act as pro-drugs which only become activated under certain conditions. Prazole, for instance, is converted at low pH from a sulfoxide to a highly reactive sulfenic acid.

specific processes. (Sulfenic acid formation is sometimes referred to as S-hydroxylation, which is formally correct yet (redox) mechanistically somewhat imprecise.) Here, oxidative, cysteinemodifying processes in the cell proceed gradually from the more reducing, accessible, and hence more reactive cysteine residues toward the less reactive ones. This kind of selectivity in oxidation is due to a range of factors, including the reactivity of the thiol (which is often associated with its pKa value due to the fact that thiolate groups are usually more reactive than thiols) and its location (readily accessible cysteine residues are often modified first). Within this context, it is noticeable that the active-site cysteine residues in enzymes are generally also the most reactive and hence also the most sensitive toward oxidation. As a result, these residues are not only prime targets for modifications, but because of their importance for function and activity, also ensure that such modifications result in a significant biochemical outcome. The modification of discrete residues in specif ic proteins subsequently enables the cell to sense changes in the cellular redox state and to provide a measured response according to the

related to the fact that cysteine modifications, which traditionally have been considered as accidental and of little consequence, appear to be more widespread in biological systems than previously thought and exert a considerable influence on cellular processes, including cellular signaling, proliferation, differentiation, and apoptosis. Indeed, the field of posttranslational cysteine modifications and conditions and reagents responsible for such modifications, has flourished during the last few years. Within this context, cysteine hunters, such as Philip Eaton, Leslie Poole, Jakob Winther, Pietro Ghezzi, Kate Carroll, and others have identified numerous mammalian and human proteins which under specific conditions, such as oxidative stress (OS), contain modified cysteine residues (Table 1).1−14 At the same time, the underlying chemistry and biochemical impact of such modifications has slowly become apparent. We now know, for instance, that most of the modifications in question, i.e., S-thiolation, S-nitrosation, and sulfenic acid formation, do not occur randomly but are the result of rather controlled, protein-specific and, in the context of structure, site589



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Table 1. Examples of Posttranslational Cysteine Modifications, Which Have Recently Been Found in Vivo, in Cells, Cellular Extracts, or in Vitroa possible cysteine modification

chemical structure

thiol disulfide S-glutathiolation S-nitrosation thiyl radical sulfenic acid

RSH RSSR RSSG RSNO RS• RSOH

sulfinic acid

RSO2H

sulfonic acid

RSO3H

thiosulfinate thiosulfonate

RS(O)SR RSO2SR

formation/ reversibilityb mild ox.c/ reversible ox./ reversible ox., •NO/reversible ox./ reversible ox./ reversible strong ox./ mostly irreversible strong ox./ apparently irreversible ox./ reversible ox./ partly reversible

trapping agent

detection method (examples only)

DTNBe reduction to thiol, then thiol specific reagent reduction to thiol, then thiol specific reagent reduction with ascorbate to thiol, then thiol specific reagent suitable spin-trap reduction with arsenite to thiol, then thiol specific reagent; or dimedone; or ICDID; or Yap1 diazonium salt (Fast Blue BB)

spectrophotometry spectrophotometry, spectrofluorometry electrophoresis, MS, confocal microscopy electrophoresis, MS, confocal microscopy EPR various techniques, including electrophoresis, LC/MS, LC-MS/MS spectrophotometry

n.a.d

n.a.

DTNB or 4-mercapto-pyridine n.a.

spectrophotometry n.a.

a

These modifications highlight the diversity of biological cysteine chemistry. One should note that the identification and analysis of such modifications is still rather difficult. It is therefore not possible at the moment to provide a comprehensive list of redox controlled cysteine proteins, their redox properties, and the subsequent impact of such modifications on cellular processes such as metabolism, proliferation, differentiation, signaling, regulation, and cell death. bReversibility is often due to the involvement of specific proteins and enzymes, such as the glutaredoxin and thioredoxin systems for the reduction of disulfides, glutaredoxins for deglutathiolation, and sulfiredoxin for the reduction of sulfinic acids to sulfenic acids. cox. = oxidant. dn.a. = a reliable analytical method is not yet available.1−14,122 eDTNB = 5,5′-dithiobis-(2-nitrobenzoic acid).

extent of redox changes. Such a response may, for instance, involves the activation of various antioxidant defenses in the case of mild OS, or it may lead to the induction of apoptosis in the case of a more severe challenge (see section 5). Since most of the cysteine modifications discussed here are also reversible, the cellular responses associated with such modifications do not have to follow a one-way road, but may rather represent cellular signaling and regulation, for instance in the form of redox feedback loops. In this respect, cysteine modifications such as S-thiolation, S-nitrosation, and sulfenic acid formation do not differ per se from other posttranslational protein modifications, such as phosphorylation, acetylation, prenylation, ubiquitinylation, ribosylation, methylation, or sulfation. Indeed, it now appears that the cellular thiolstat, i.e., the complex network of (redox) regulated cysteine proteins and enzymes, forms a key element in cellular signaling which interferes with various other cellular signaling pathways and ultimately contributes to essential life and death decisions. While the thiolstat as a cellular rheostat is primarily based on thiol modifications, one should point out that other cysteine based modifications beyond the thiol state, such as sulfenic/sulfinic acids, also play a role in regulation and signaling.

reactivity is clearly not desired, and identifying agents which also react with specif ic target proteins in the presence of a wealth of other cysteine proteins and millimolar concentrations of reduced glutathione (GSH) is an extremely challenging task. Anyone determined to target cysteine residues in key proteins and enzymes therefore has to heed a few basic lessons. Thiol groups in general are quite good electron donors as well as nucleophiles (which react readily with soft electrophilic species) and hence are often rather prone to oxidation. Therefore, strong oxidants may be useful to trigger widespread oxidation of cysteine residues (and subsequent cell death) yet are hardly suitable as selective agents to target specific cellular pathways. Most reactive oxygen species (ROS), for instance, react with and subsequently inactivate a range of cysteine containing proteins and enzymes, yet they are often too aggressive to target specific proteins. While such agents may qualify as hard-hitting pesticides or may be suitable as sterilizing agents (such as bleach), they clearly cannot be used for more subtle applications, e.g., as topical or even systemic medications. Interestingly, as a good nucleophile, the thiol group reacts particularly well and frequently (but not always) also selectively with electrophilic agents based on oxygen, sulfur, selenium, and tellurium. This special relationship is exemplified by the human glutathione peroxidase (GPx) enzymes: the latter employ an active-site selenocysteine residue to catalyze the reaction of peroxides with GSH to water/alcohols and GSSG, and the most effective GPx mimics are based on selenium and tellurium.15−17 This selectivity may be explained in part by the specific redox mechanisms which underlie thiol-modification reactions. Most of these reactions do not proceed via electron transfer but other mechanisms, such as radical reactions, atom transfer, and nucleophilic exchange reactions (Figure 2). In the context of selectivity, exchange reactions are of particular interest since thiols and disulfides seem to react efficiently in the form of a socalled thiol/disulfide exchange. Indeed, there are numerous examples of this exchange reaction in biology, often involving GSH.16,18 Since disulfides react efficiently with thiols and also show a high degree of selectivity for the thiol nucleophile over other

2. INTERFERING WITH CYSTEINE PROTEINS As the number and diversity of cysteine containing proteins known to be under redox control increases, we are becoming more aware of the fact that modifying cysteine residues in proteins and enzymes may provide a promising target to interfere with a wide range of cellular processes. Despite the strong desire of biochemists and pharmacologists to control cysteine-centered signaling, however, there are some obstacles which need to be overcome, especially in the context of selectivity. Ultimately, this selectivity is 3-fold: in the first instance, it includes selectivity for thiol groups, followed by selectivity for specific cysteine proteins and ultimately for specific cysteine residues within these proteins. While most cysteine-modifying agents considered in this context today are specific for thiols, they still react more or less randomly with any thiol present inside the cell. This kind of unspecific 590



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environment. Thioredoxin (Trx) proteins, for instance, contain cysteine residues with a potential of −270 mV (redox potential) vs NHE, while the active site cysteine residues in protein disulfide isomerases (PDIs) are considerably more oxidizing, with a potential of −190 mV (reduction potential).16,20 As already mentioned, such differences in electrochemical potential, and hence susceptibility toward oxidation, provide a certain selectivity for thiol-specific agents which usually react preferably with the more reducing and the more accessible thiol groups. Nonetheless, one must emphasize that the Epa values are not the only determinants of oxidative modification of cysteine residues in proteins and enzymes. The location and hence the accessibility of cysteine residues (e.g., at the surface or buried within the protein), as well as the location of protein as a whole (e.g., cytosolic, nuclear, and membrane bound) and proximity to the modifying agent are equally important.21 Within this context, recent research has considered several chemically rather interesting compounds, whose unique properties deserve our close attention. Among these agents, we find substances able to trigger a wider shift in the cellular redox state E as well as compounds which only modify the most reactive cysteine residues in proteins. Furthermore, sulfur chemistry lends itself to the design of pro-drugs, some of which are activated enzymatically, while others are being transformed into their active form as a result of low pH. We will now discuss some of the rather exquisite chemistry behind such agents and subsequently highlight prime targets of such modifications, focusing on proteins controlling the cell cycle and initiating cell death. While we cannot provide a complete or ultimate overview of all relevant cysteine modifying agents available to date, we will highlight a selection of promising agents which deserve our close attention in the near future.

Figure 2. Thiols are able to undergo redox transformations by a range of different mechanisms. Besides one- and two-electron transfer, hydrogen atom abstraction, and radical reactions, thiol/disulfide exchange reactions play a particularly important role. The inherent flexibility in reaction mechanisms, and hence potential reaction partners, endows thiols (and other sulfur species) with a facet-rich redox chemistry in vitro and in vivo. Note that this scheme of possible reactions is not exhaustive as a number of less commonly encountered mechanisms are not shown.

nucleophiles (e.g., alcohols and amines), disulfide-based agents have been at the forefront of the development of cysteinespecific modifying agents. Here, the natural disulfide diallyl disulfide (DADS), which occurs in garlic and related Allium plants (like onions), may serve as an example (Figure 1).19 This compound appears to oxidize, i.e., S-thiolate, a range of key proteins and enzymes and is currently under discussion for possible medical and antimicrobial applications. Nonetheless, DADS also highlights the dilemma of many thiol-specific agents which are selective for thiols yet unspecific for individual cysteine residues. Such agents modify various peptides and proteins in the cell, including GSH, and most of them react rather slowly with thiols in the absence of suitable catalysts. Ultimately, a large excess of disulfide (up to millimolar concentrations) may therefore be required to achieve the desired effect within a reasonable period of time, which in turn reduces selectivity. In order to target cysteine proteins more effectively, one must therefore turn toward agents that are more reactive than DADS yet, at the same time, are not too reactive as to modify cysteine residues indiscriminately or to become sequestered by GSH (see above). At this point, the issue of thiol oxidation potentials (Epa) turns out to be of paramount importance. While most reports refer to the redox potential E0 of a given cysteine residue in a particular protein, we prefer to focus on the oxidation potential Epa. The latter describes more appropriately the aspects of the initial, oxidative thiol modifying process, hence avoiding questions related to reversibility of the modification and aspects of its subsequent reversal, for instance by reduction. The question of reversibility of a given modification is important, of course, yet often leads to rather complicated processes involving different agents, enzymes, and even reaction mechanisms. In those cases, the redox potential E0 is no longer an appropriate descriptor of the overall process. Nonetheless, one must note that the values currently available in the literature reach from oxidation potentials to redox potentials and reduction potentials (Epc) and often cannot be compared directly with each other. Similar to the pKa values, the Epa values of different cysteine thiols in the cell vary quite considerably. The thiol group in GSH, for instance, has a reduction potential between −230 and −240 mV vs the normal hydrogen electrode NHE (the precise value depends on the literature sources considered and the analytical methods employed therein). This value differs from the ones found in many cysteine proteins, where values of individual cysteine residues depend on their protein micro-

3. FROM SIMPLE DISULFIDES TO HIGHLY REACTIVE SULFUR SPECIES Among the various agents which react readily with thiols, we find several molecules that may be considered as activated forms of the disulfide motif. Two of the simplest and at the same time most reactive disulfide derivatives are thiosulfinates and thiosulfonates (Figure 1). In essence, thiosulfinates and thiosulfonates initially react with thiols similar to a disulfide, yet are considerably more reactive because one of their sulfur atoms is oxidized. The natural garlic ingredient allicin (2-propene-1sulfinothioic acid S-2-propenyl ester), for instance, is formed chemically by the oxidation of DADS. Interestingly, the biosynthetic pathway of allicin does not proceed via the oxidation of DADS. Allicin is rather formed from its sulfoxide precursor alliin in an enzymatic reaction. The latter involves the enzyme alliinase and proceeds via the cleavage of a carbon− sulfur bond, formation of a sulfenic acid, and subsequent dimerization of two of these sulfenic acids to allicin and water. Despite its close structural similarity with DADS, its reactivity toward thiols is orders of magnitude higher. This change in reactivity when moving from the disulfide to the disulfide-Soxide(s) is due to a dramatic increase in electrophilic properties of the sulfur atom and a pronounced follow-on chemistry which opens up once the thiosulfinate is reduced (Figure 3). While most authors discuss the reactivity of the various reactive sulfur species in a more qualitative or semiquantitative manner, Michael T. Ashby and colleagues have recently published a series of kinetic studies on reactive sulfur species, including one on cysteine thiosulfinate esters.22−25 591



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Figure 3. The thiosulfinate allicin is a highly reactive sulfur species. In garlic, it is formed from its sulfoxide precursor alliin in an enzymatic reaction involving the C−S-lyase alliinase. It reacts efficiently with thiol groups, hence modifying proteins and enzymes and triggering a follow-on chemistry involving a reactive sulfenic acid and various disulfides. It also decomposes to form a range of other reactive sulfur species, including diallylsulfide (DAS), DADS, DATS, and DATTS. (Pr = protein).

e.g., inside the cell.26 Prominent examples of drugs which react via a transiently formed sulfenic acid are the prazole protonpump inhibitors, such as Omeprazole,29 which are employed in the treatment of stomach ulcers (Figure 1). These compounds exhibit a rather interesting chemistry, which in fact resembles some aspects of the alliinase enzyme chemistry mentioned above. Like alliin, prazoles are pro-drugs which contain a sulfoxide moiety. Under acidic conditions (e.g., in the stomach), a carbon−sulfur bond of the prazole is cleaved and the resulting molecule rearranges to form a sulfenic acid (Figure 4). The latter then reacts with a critical cysteine thiol of H+/K+-ATPase, subsequently inhibiting the enzyme and raising the pH value in the stomach. Since the rearrangement to the active form is acidtriggered and therefore critically depends on the pH value of the medium, sulfenic acid formation subsides once the pH increases. Together with pH activation, this feedback endows prazoles with considerable selectivity. First, the sulfenic acid is only generated in cells lining the stomach wall, while other parts of the human body, which exhibit considerably higher pH values, are not affected. Second, the transformation of the sulfoxide to the sulfenic acid cedes once the pH value of the stomach reaches therapeutically desirable values. The prazoles illustrate the powerful, diverse, and often intelligent biological chemistry of reactive sulfur species, which

Widespread S-thiolation of cysteine residues in proteins and enzymes, either by allicin directly or by modified forms of GSH (such as S-allylmercaptoglutathione) more indirectly,26 appears to be responsible for the pronounced cytotoxic properties which this and related thiosulfinates exhibit against various microbes and certain human cells.27,28 In addition, the sulfenic acid liberated as part of this reaction reacts efficiently with thiols under the formation of a mixed disulfide (which may react further) and water. Depending on the reaction partners and conditions present, the mixed disulfide formed as part of this initial reaction may even react further with another (protein) thiol to form a more stable disulfide and the fully reduced thiol (e.g., allylmercaptan); yet this reaction (i.e., a classical thiol/ disulfide exchange) is generally considerably slower than the one involving the thiosulfinate (or sulfenic acid). Besides thiosulfinates and thiosulfonates, sulfenic acids, which occur quite frequently in proteins and enzymes under conditions of OS, have recently attracted considerable interest. Sulfenic acid species are able to S-thiolate cysteine residues rather efficiently. Nonetheless, most of them are not suitable as drugs since they are chemically unstable and react with oxidants, such as dioxygen, to form more stable sulfinic or sulfonic acids. It is possible, however, to employ certain sulfenic acidprecursors which are only activated at or near the target site, 592



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Figure 4. Activation of prazole is a fine example of a sulfoxide-to-sulfenic acid conversion, which bears considerable potential as far as pro-drug activation and reactive sulfur species are concerned. Activation of the sulfoxide-containing prazole prodrug to the active sulfenic acid is controlled by pH. It may involve a less reactive sulfenamide, which in itself is an interesting sulfur species in the context of drug design.

simply combine to a disulfide. This kind of reaction requires the involvement of an oxidizing agent or the initial oxidation of one (or both) of the two thiols involved. Interestingly, a recent report has postulated the involvement of a transiently formed sulfenic acid, which has been trapped with the assistance of thiols and the sulfenic acid-specific reagent dimedone.38 Compared to conventional disulfide-based reagents, disulfide-S-oxides and sulfenic acids have certain advantages, especially in the context of reactivity; most of them, however, are too reactive and therefore not particularly selective for specific cysteine residues in particular proteins. As already mentioned, a random modification of cysteine residues in peptides, proteins, and enzymes may be useful in the context of antimicrobial defense (e.g., antibiotics, fungicides, and nematicides), yet a more selective action is required in order to target specific cellular signaling pathways. Polysulfanes, such as diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic, are considerably less reactive and hence more specific compared to allicin (Figure 1). So far, several proteins have been identified which appear to represent (some of) the prime targets of DATS, including β-tubulin and Keap-1 (DATTS is likely to react in a similar fashion, yet less is known about this compound to date).39,40 Nonetheless, the precise chemical reaction(s) and biochemical mode(s) of action associated with DATS and DATTS are still not fully understood. Besides initial S-thiolation of proteins and enzymes, it appears that the resulting reduced forms, most likely perthiols (RSSH, R ≠ H) and hydropolysulfanes (RSxH, x ≥ 3, R ≠ H), may undergo a range of biochemically interesting follow-on reactions, including ROS formation, Sx and Sx2− release, and interactions with metal ions in metalloproteins (see section 5). Of notable interest from a biochemical perspective are radical generation (via the reduction of dioxygen), Sx release, and H2S release reactions associated with polysulfanes and their reduced forms.41−44 At the same time, polysulfanes are not only redox

includes selective activation and feedback loops. They also showcase the transformation of a sulfenic acid to a less reactive sulfenamide, a rather unusual sulfur chemotype which has been observed recently in enzymes, such as protein−tyrosine phosphatase 1B (PTP1B).30−32 In drug design, sulfenamides may act as useful precursors or even alternatives to the more reactive sulfenic acids. In any case, the prazoles underline the claim that transiently formed sulfenic acids may be used as cysteine-modifying drugs in vivo. Here, these compounds clearly provide a blueprint for future drug design, whereby fairly unreactive sulfoxide-based prodrugs may be activated to form highly reactive sulfenic acids by cleavage of one of the carbon− sulfur bonds as the key step of activation. Interestingly, cleavage of the carbon−sulfur bond can be facilitated chemically (as in the case of prazoles) or enzymatically (as in the case of alliin). Enzymatic activation opens the door to highly selective twocomponent drug and pesticide systems which have hardly been explored to date.28 A similar sulfur-centered activation of a pro-drug in response to a particular physiological process, this time the presence of certain enzymes, is observed in the case of the tetrahydrothienopyridine-based antithrombolytic drug Clopidogrel (Figure 1).33 In this case, the initial tetrahydrothienopyridine moiety is not particularly reactive on its own. Hydroxylation by certain cellular oxidases, such as cytochrome P450 2C19 (CYP2C19), however, results in a thiolactone, which is subsequently hydrolyzed to an acid and a thiol (Figure 5).26,34 The latter reacts with a particular cysteine residue of the ADP-receptor P2Y12 to form a disulfide, a modification of P2Y12 which ultimately inhibits the aggregation of blood platelets.35−37 The precise chemical events behind the modification of P2Y12 are still not fully understood, and the literature is often elusive on the exact processes leading to the formation of the disulfide. Obviously, the two thiol groups in question, i.e., the thiol belonging to the hydrolyzed form of oxidized Clopidogrel and the cysteine residue in P2Y12, do not 593



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Figure 5. Oxidative activation of the thrombolytic drug Clopidogrel. Previous studies have reported the enzymatic oxidation of the tetrahydrothienopyridine to a thiolactone. The latter is hydrolyzed to release a reactive thiol which then modifies a cysteine residue of the target protein, i.e., the ADP-receptor P2Y12. While the precise chemistry of this modification is still unclear, a recent report has indicated the possible involvement of a transient sulfenic acid, which could be trapped by thiols and the sulfenic acid specific agent dimedone.

opens up an intricate follow-on chemistry, probably involving a whole cascade of biologically relevant, reactive species: initially, an α,β-unsaturated thiol is formed, which may rearrange to a highly reactive thioaldehyde (Figure 6A). Although speculative at this time, the latter may react with thiols (and other nucleophiles, e.g., amines) in order to modify additional proteins. Products of such reactions may include further (re)active compounds, such as a dithioacetal and aminothiol or a disulfide and sulfenamide. This cascade of reactive products may explain why 1,2-DT exhibits good antimicrobial activity in certain biological assays and increasingly places this and related α,βunsaturated disulfides into the focus of drug design and pesticide development. Ajoene also exhibits a broad spectrum of antimicrobial (and chemopreventive) activity, which is mainly attributed to the motif of a disulfide combined with a vicinal double bound.49,50 A similar behavior has been found in the case of thiuramdisulfides, which exhibit considerable reactivity toward thiols and possess a particular follow-on chemistry. The compound disulfiram (1,1′,1″,1‴-[disulfanediylbis(carbonothioylnitrilo)]tetraethane, tetraethylthiuramdisulfide), for instance, is a highly reactive, thiol-specific reagent, which in the past has been used under the name Antabuse to treat alcoholism (Figure 1).51,52 Disulfiram metabolites react with key cysteine residues in the human target enzyme aldehyde dehydrogenase, which is subsequently inhibited (Figure 6B). In the presence of ethanol, this particular inhibition results in an accumulation of acetaldehyde in the body and a range of unpleasant physiological effects. The latter are thought to reduce one's inclination to consume alcoholic beverages. The reduced form of the thiuramdisulfide, i.e., the dithiocarbamate, is a rather interesting chemical species on its own, whose reactivity may not be limited to redox processes, but may also involve interactions with metal ions, for instance at the active site of metalloproteins. Although still speculative, such dithiocarbamates may possibly also release the signaling molecule hydrogen sulfide (H2S) under physiological conditions,

active but also bind to metal ions and are hydrophobic, i.e., they can interact with membranes and hydrophobic pockets of proteins and enzymes. These interactions can take place in addition to the redox reactions or as separate processes, for instance in cells that lack an extended redox network, such as red blood cells.45 One should point out that DATS and DATTS are not particularly exotic molecules as far as biology is concerned. There are several other (natural) polysulfanes of therapeutic interest, including the pentasulfane-containing marine product varacin (2-(6,7-dimethoxy-1,2,3,4,5-benzopentathiepin-9-yl)ethanamine), which is found in marine Ascidiacea. This substance exhibits pronounced antibacterial and DNA-damaging activity and has recently received attention in the context of antimicrobial and anticancer activity. 42,46 Furthermore, the hexasulfane lenthionine (1,2,3,5,6-pentathiepane), which is found in Shiitake mushrooms (Lentinus edodes), has also attracted some interest as a potential antimicrobial or anticancer agent.47 As far as natural sulfur products are concerned, garlic is a true treasure chest of unusual compounds, of which some exhibit considerable biological potential. In this context, α,β-unsaturated disulfides, such as ajoene and 3-vinyl-3,4-dihydro-1,2dithiin (1,2-DT), stand out. It should be mentioned that several organic isothiocyanates also occur naturally in edible plants, such as broccoli, and have recently been considered as potential prototype drugs. While these isothiocyanates do react with cysteine residues in proteins, they also react with various amines and hence are rather nonselective, at least from a chemical point of view.47,48 Like allicin, ajoene and 1,2-DT have formed part of the human diet for several millennia and provide a lead for therapeutically active agents, which appear to be largely nontoxic for humans. Because of the unsaturation present in the vicinity of the disulfide bond, 1,2-DT appears to be considerably more reactive than a normal disulfide. Furthermore, the initial thiol/disulfide exchange reaction 594



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Figure 6. α,β-unsaturated disulfides such as 1,2-DT seem to be fairly reactive toward thiol groups, possibly because of the ability of the reaction products to rearrange to a thioaldehyde and hence react further with thiols and other nucleophiles (A). From the perspective of biological chemistry, the thiuram disulfide chemotype is particularly interesting since its reaction with a given thiol results in a thiocarbamate, which may interact further with proteins, for instance by blocking catalytic metal sites (B). Such compounds may also release the signaling molecule H2S.

hence liberating the transcription factor Nrf2 from the Keap-1Nrf2 complex.40,56−58 Once liberated, Nrf2 translocates to the nucleus where it then initiates the expression of antioxidant enzymes. One should note that the chemistry underlying the oxidation of Keap-1 is not yet fully understood: the 1,2-dithiole3-thione may oxidize certain key cysteine residues of Keap-1 directly, or indirectly.56 Nonetheless, Keap-1 is a prominent target in cancer research, where Nrf2 plays a role as part of chemoprevention as well as in therapy (see also section 5).57,58 Like Oltipraz, anethole dithiolethione, which is already used in therapy as cholereticum, has also been associated with (indirect) antioxidant activity, possibly by inducing the synthesis of GSH and/or by up-regulating Phase 2 enzymes. During the last decades, anethole trithione has been the object of intense biochemical studies. Apart from its activity as cholereticum, and suspected antioxidant activity, the compound has also been considered, for instance, as an inhibitor of NF-κB activation in human T cell lines and as a regulator of oxidantinduced tyrosine kinase activation in endothelial cells.59−61 In order to clarify the biochemical mode(s) of action of such compounds, one needs to investigate the chemistry of this

for instance via hydrolysis. Not surprisingly, disulfiram and its derivatives, which have been used in medicine in the past, are currently experiencing a renaissance among pharmaceutical chemists, and are being investigated as potential antimicrobial and anticancer agents.53,54 Another sulfur chemotype related to the disulfide is the 1,2dithiole-3-thione, which contains a reactive thioacyldisulfide moeity (Figures 1 and 7). From a chemical point of view, 1,2dithiole-3-thiones can be formed rather easily, for instance by reacting compounds containing one or more carbon−carbon double bonds with elemental sulfur (S8). Indeed, there are reports that certain natural remedies also contain 1,2-dithiole-3thiones, among other substances. Some 1,2-dithiole-3-thiones, such as Oltipraz (4-methyl-5-(2-pyrazinyl)-3-dithiolethione) and anethole dithiolethione (Sulfarlem, 5-(4-methoxyphenyl)3H-1,2-dithiole-3-thione), are even used as drugs. Oltipraz is a powerful schistosomicide (i.e., an agent effective against parasitic worms) and is often also considered as a potential antioxidant.55 The apparent antioxidant action, however, appears to be mostly indirect: rather than acting as a reducing agent, Oltipraz is likely to react as a weak oxidant, oxidizing the protein Keap-1 and 595



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dithiole-3-thione chemotype still hides many chemical and biochemical secrets, and its special reactivity is of considerable interest in drug design and antimicrobial agent/pesticide development.63−65 Several of the examples discussed so far have highlighted the important role that initial activation steps and follow-on reactions play in the context of effective, yet selective cysteine modification. Within this context, compounds such as Clopidogrel and the prazoles support the notion of pro-drugs, which become activated only under certain conditions or at the target site. At the same time, thiosulfinates, thiosulfonates, thiuramdisulfides, and 1,2-dithiole-3-thiones illustrate how sulfur chemistry can form the basis for whole cascades of transformations and modifications, all of which can occur under cellular conditions. We will conclude this section with a brief discussion of an emerging redox-system, which shows some unusual selectivity for cysteine proteins and connects sulfur with reactive nitrogen species. Benzothiazinones (BTZs) have been studied recently as possible antituberculosis agents.66 It has now become apparent that these BTZs develop their antimycobacterial activity by attacking an essential cysteine residue in the enzyme decaprenylphosphoryl-β-D-ribose 2′-epimerase (DPRE), which is inhibited by this modification. Interestingly, the BTZ agents, which contain a nitroarene function, themselves are not reactive toward thiols. Such compounds, however, become activated by the reduction of the nitroarene to the more reactive nitrosoarene. The latter reacts with thiols to form a rather unusual N-hydroxysulfenamide, which may react further with thiols to form a disulfide and hydroxylamine; the N-hydroxysulfenamide may also rearrange to a more stable sulfonamide (Figure 8).67,68 In this particular case, the combination of (bio)reductive activation and cysteine-specific reactivity appears to endow the benzothiazinones with considerable selectivity, especially for the DPRE enzyme. While the underlying chemistry and biochemical impact of these agents is only just emerging, they open up several new avenues for the design of cysteine-specific agents and bioreductive drugs (e.g., for cancer therapy).

Figure 7. 1,2-Dithiole-3-thiones provide many exciting opportunities for drug design and pesticide development. Such compounds are formed chemically rather easily and exhibit a usual, yet biologically rather interesting reactivity and follow-on chemistry (see also Figure 6). Not surprisingly, 1,2-dithiole-3-thiones have been found in some remedies and also provide the basis for several drugs, including in the schistosomicide Oltipraz and the choloreticum anethole dithiolethione.

particular chemotype once placed inside a living cell. At first sight, the 1,2-dithiole-3-thione moiety seems to combine aspects of an α,β-unsaturated disulfide with an α,β-unsaturated thione. As far as we know, such an agent may react with a given thiol in different ways, depending on the reaction partners and conditions present (Figure 7). Several years ago, Fleury et al. studied the Oltipraz chemistry in considerable detail. Besides interactions with amines, alcohols, and dioxygen, the reactions with thiols were of particular interest.62 Possible interactions with target proteins, some of which are clearly speculative at this time, include attack of a cysteine thiol at the disulfide function of the 1,2-dithiole-3-thione with subsequent formation of a mixed disulfide and opening of the five-membered ring. Depending on the position of attack, a mixed disulfide and either an α,β-unsaturated thiol or a dithiocarboxylate are formed by this process. The mixed disulfide may react further with another thiol, while the vinylic thiol may rearrange to form a highly reactive thioaldehyde (see above). The thiocarbamate, in contrast, may interact with metal ions or possibly even release H2S. Indeed, H2S release from dithiolethiones (or their reduced forms) may explain some of the antioxidant as well as cytotoxic properties associated with such compounds. Baskar et al. have recently studied the inhibitory effects of S-diclofenac on rat vascular smooth muscle proliferation and have attributed the effects observed to H2S release from this particular dithiolethione.61 One should also note that the carbon−carbon double bond in the 1,2-dithiole-3-thione may provide a site for a Michael-type nucleophilic attack, not only by thiols but also possibly by other nucleophiles. Since many of the follow-on products of the initial reaction(s) of the 1,2-dithiole-3-thione are also highly reactive, 1,2-dithiole-3-thiones may exhibit a complex behavior inside the cell, possibly affecting various targets in different ways. Here, one should also note the possibility of oxidative elemental sulfur release from 1,2-dithiole-3-thiones, such as Oltipraz, during metabolism.62 Ultimately, the 1,2-

4. TECHNIQUES TO PROBE THE REACTIVITY OF CYSTEINE AND ITS POSTTRANSLATIONAL MODIFICATIONS The last section has shown that various different classes of substances exhibit a high and often rather selective reactivity for thiol groups in proteins and enzymes, at least in vitro. Not surprisingly, this raises the question which proteins/enzymes are affected primarily by such agents in vivo. As already mentioned, a specific reactivity for particular proteins, in addition to a selectivity for the thiol group in general, may be extremely useful for targeting individual proteins and pathways associated with them. Thus, it is easy to conceive that much effort has been dedicated to the development of biochemical tools for the identification and/or quantification of reactive thiols as well as their posttranslational modifications. Such tools are indeed pivotal elements to gain insight into cysteine-based and redoxdependent biochemical and toxicological events. We provide here a nonexhaustive list of some of these tools currently available, summarized in Table 1 (for a more comprehensive recent review, see for instance Leonard and Carroll69 and references therein). Reactive cysteine residues, either as free thiols or engaged in intra- or intermolecular disulfide bridges, can be classically 596



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Figure 8. Reductive activation of benzothiazinones. The conversion of the nitroarene to the nitrosoarene results in a highly reactive nitrogen species which is able to modify essential cysteine residues in the target protein decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE). The chemistry of this modification reaction is rather interesting; it involves a semimercaptal and sulfonamide.

specific algorithms, such as a “procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases.”74 Such emerging computational approaches bear considerable promise, also for the ranking of (re)activity, and have been reviewed recently by Marino et al.73 Several methods are available for the detection of sulfenic acid modifications in proteins and enzymes.75 Cysteine sulfenic acid can be detected in purified proteins using the sulfenic-acid specific probe 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl). Interestingly, while NBD-Cl reacts with both, cysteine thiols and cysteine sulfenic acids, the resulting adducts differ considerably in their spectrophotometric properties. λmax of NBD adducts with cysteine thiols is observed at 420 nm, while the sulfenic acid-NBD adduct has a maximal absorbance at 347 nm.76 The major drawback of such a method, however, resides in the fact that it requires purified proteins, i.e., only works in vitro. Here, a major advantage of selectively targeting cysteine sulfenic acids with dimedone derivatives is the possibility of further tagging the chemically rather simple dimedone core with more specific and innovative additions.12,13 Some of these sulfenic acid specific dimedone reagents are nowadays even commercially available. Such dimedone-based probes can be advantageously used on complex protein mixtures, thus providing an overall picture of the transient sulfenic acid proteome.77 Within this context, a rather elegant method of mapping out the cellular sulfenome, employing isotope-coded dimedone and iododimedone (ICDID), has recently been developed by Kate Carroll and colleagues.12,13 The same group has also just reported a series of rather innovative probes for protein tyrosine phosphatases (PTPs), which combine dimedone warheads with a specific binding module (for PTP) and an azide reporter tag for downstream

cross-linked with a panel of reagents such as maleimide or iodoacetamide coupled to detection tags.70 For instance, a method has recently been described for measuring both reduced and oxidized cysteine residues in proteins whereby reduced thiols are first labeled with a fluorescent BODIPY maleimide derivative.71 Disulfide bridges are then reduced and labeled with a Texas Red maleimide derivative, allowing for the simultaneous detection of reduced protein thiols and disulfides. While this method allows the quantification of thiols and disulfides inside the cell, it does not (easily) identify which individual proteins have been modified and to which extent. Here, a major step forward in the identification and quantification of particularly reactive cysteine residues in the native proteome has recently been provided by Weerapana et al.72 The method, which the authors call isoTOP-ABPP (isotopic tandem orthogonal proteolysis-activity-based protein profiling), is based upon the use of a cysteine-directed iodoacetamide probe, the resulting adduct bearing a TEV protease recognition peptide tagged with biotin and labeled with either a light or heavy 13C-labeled valine. After binding to a streptavidin column, proteins are released upon incubation with TEV protease, digested with trypsin and analyzed by mass spectrometry. It is then possible to assess the extent of labeling by comparing high versus low iodoacetamide probe concentrations and to score cysteine reactivity in proteins. Overall, the method allows for the detection, quantification, and classification of the whole proteome by mass spectrometry according to cysteine reactivity. Interestingly, there have also been several rather promising attempts to classify cysteine residues in proteins and enzymes by various bioinformatics approaches, which has allowed the identification of some of the most reactive and regulatory residues.73,74 These in silico methods employ a number of 597



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analysis.78 Since PTPs play a major role in intracellular control and signaling and appear to possess a particularly reactive active site cysteine residue, such probes are of considerable interest to the cell signaling community.12,78,79 They may also provide valuable leads for the design of specific PTP inhibitors that target the sulfenic acid (rather than the thiol) form, which is found in these enzymes.78 Protein glutathiolation can occur when a cysteine sulfenic acid comes into contact with the most abundant nonprotein thiol glutathione (GSH). S-Thiolation is reversible, especially in the presence of glutaredoxins (Grx) and, together with sulfenic acid modification, is emerging as an exquisite regulatory mechanism impacting on various redox-dependent cellular signaling pathways.80 In general, protein S-glutathiolation can be detected using commercially available anti-GSH antibodies. A number of alternative methods employ a sequence of capping unmodified thiols, subsequent reduction, and tagging the reductively liberated thiols. Some of these methods have recently been reviewed.81 As an example, the method reported by Aesif et al. serves the in situ detection of S-glutathiolated proteins.82 As part of this technique, free protein thiols are first alkylated by N-ethylmaleimide (NEM) in order to quench their reactivity. This is followed by Grx1-catalyzed deglutathiolation of S-glutathiolated residues and reaction of the liberated thiols with a biocytin-maleimide derivative. Ultimately, these residues are detected using streptavidin-conjugated fluorophores and confocal microscopy. Nitrosation of cysteine residues in proteins is another example of a reversible modification found in cells as part of a control mechanism governing many aspects of cellular functions. Recent progress in the study of protein nitrosation and visualization of this posttranslational modification is based on the use of antibodies that recognize S-nitrosocysteine.83 Biotin-switch methods such as the one described by Aesif et al.82 are currently available for the detection of in situ nitrosated proteins. As part of this method, proteins are incubated with NEM in order to alkylate free thiols. Nitrosothiols are then reduced with ascorbate, and the resulting free thiols are labeled with a biocytin-maleimide derivative combined with a fluorescent streptavidin as described above for protein S-glutathiolation. In addition to ROS and nitric oxide synthase (NOS)mediated posttranslational modifications, electrophilic compounds such as the byproducts of redox reactions as well as xenobiotics and their metabolites can react with cysteine thiol(ate)s and impact both on the function of proteins as well as on cellular signaling pathways. Compounds with an electrophilic β-carbon, for instance, are prone to react with nucleophilic thiolates via Michael addition.84 Within this context, α,β-unsaturated carbonyls (acrolein, 4-HNE, and 15-deoxy-Δ12,14-prostaglandin J2) have been reported to inactivate the phosphatase tensin homologue on chromosome 10 (PTEN)85 and consequently increase cell proliferation. PTEN alkylation occurs through a particular cysteine modification and can be detected using a neutravidin−biotin pull-down method: cellular proteins (including PTEN) with either oxidized or carbonylated thiols are first alkylated with NEM or iodoacetic acid in order to modify any free thiols prior to reduction of oxidized or carbonylated residues. Once liberated by reduction, the previously modified thiols are then selectively tagged with NEM-biotin and pulled down using neutravidin beads. An immunoblot of specific proteins on the pull-down sample thus allows for the identification of oxidized or carbonylated proteins. A variation of this strategy uses a chemical probe bearing the

proper tag for easy identification. Covey et al., for instance, have used cyclopentenone prostaglandins tagged with biotin in order to identify cysteine-based Michael adducts with PTEN.85 Importantly, PTEN is not the only enzyme with a cysteine residue prone to attack by α,β-unsaturated aldehydes. Other examples include protein disulfide isomerase (PDI) and PTP 1B, both of which can be inactivated by acrolein,32,86 and SHP-1, which is inhibited by 4-hydroxynonenal.87 Click chemistry using electrophilic compounds bearing an azide handle and alkyne-biotin tag can also be applied for the detection of cysteine adducts in proteins.84 Keap-1 may serve as an example. Recently, sulfoxythiocarbamate analogues have been found to form stable adducts with Keap-1 cysteines, triggering the induction of the Keap-1/Nrf2 antioxidant response elementdependent pathway.88 Adducts with key Keap-1 cysteine residues have been identified in the same work using one of the sulfoxythiocarbamate analogues bearing an alkyne handle for convenient click chemistry-based tagging. While these methods allow the more or less specific identification of certain sulfur modifications in vitro (and increasingly also inside living cells), one must bear in mind that there is a certain selectivity in reactivity between different (modified) cysteine residues in proteins (see also section 2). Ultimately, some of the modifications may therefore be detected more readily (e.g., the ones which are more reactive and which occur at the more accessible residues), while others may be more difficult to access by the probe and hence may be harder to track down. This matter becomes important once modifications present at buried cysteine residues are concerned.

5. ASPECTS OF THE BIOCHEMICAL IMPACT OF POSTTRANSLATIONAL CYSTEINE MODIFICATIONS With the constant development of a wide panel of chemical toolkits enabling us to study cysteine reactivity and posttranslational modifications, and an increasing sophistication of proteomic methods, our understanding of the redoxome89,90 and its many roles in health and disease is likely to grow significantly over the next couple of years. At the same time, more specific modifying agents are likely to enter the fray, which promise good selectivity for specific (cysteine protein) targets. This brings us to the question, which specific biochemical processes are affected by particular cysteine modifications inside the cell and which may, for instance, be exploited in the context of chemoprevention or anticancer drug design. The answer to this question is not trivial. While research during the past decade has provided ample evidence for the widespread occurrence of cysteine modifications inside living cells, such as intra- and interprotein disulfide formation, S-thiolation (primarily Sglutathiolation), S-nitrosation, and sulfenic acid formation, it is still not fully understood how these processes are controlled and how they act (together) in order to generate an appropriate cellular response. Even so, some aspects of control via the intracellular thiolstat have recently been emerging.91,92 Under severe conditions of OS, for instance, there appears to be widespread, yet mostly reversible oxidation of cysteine residues in GSH as well as in numerous proteins and enzymes. In this case, cysteine residues act as sacrificial thiols, as a kind of redox buffer which provides a certain defense against the oxidative insult. Here, recent studies by Jakob Winther and colleagues have revealed that cysteine thiols in proteins and enzymes and not just in GSH may be the prime targets of oxidation (in this particular study by diamide).9 In any case, the impact of such widespread protein oxidation/modification is 598



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explains why the cell can respond toward redox changes in a gradual and appropriate manner, rather than via a random and all-or-nothing response. Such a gradual response is in good agreement with an earlier hypothesis of Freya Schafer and Garry Buettner.99 This model, published in 2001, links the intracellular redox state expressed by the GSSG/GSH ratio to cell proliferation, differentiation, and apoptosis. Not surprisingly, there are several examples of cysteine modifying agents which interfere with cell cycle progression, apoptosis, and differentiation. One of the most apparent and at the same time prominent targets for such agents is β-tubulin. β-Tubulin contains redox sensitive cysteine residues and can be modified by agents such as DADS and DATS, which directly oxidize, i.e., S-thiolate this protein, hence inhibiting tubulin polymerization and the formation of the mitotic spindle.39,100 In SH-SY5Y neuroblastoma cells, DADS induces significant alterations in cell morphology, including microfilament disruption and alterations in the microtubule network. While some of these effects may be due to direct interactions of DADS with β-tubulin, others may be the (indirect) result of an increase in intracellular ROS levels, which also leads to widespread oxidation of cysteine residues.39,101 There are, however, also other potential targets for such compounds inside and outside the cell, which complicates matters considerably. Here, DADS increases the level of the connexin Cx43 protein, which in turn improves gap-junctional intercellular communication.102 So far, it still remains to be seen how the connexin Cx43 level is influenced by DADS, but a redox process involving a cysteine protein in charge of Cx43 levels appears likely. Furthermore, DADS has an effect on H3 and H4 histones, whose acetylation increases significantly in the presence of this compound, possibly because DADS inhibits histone deacetylase (HDAC) enzymes.103 It is still unclear if this inhibitory effect is related to a redox process or rather involves coordination of DADS or its reduced form, allyl mercaptan, to the zinc ion at the HDAC active site.104 In any case, changing the degree of histone acetylation has a major influence on gene expression, and some effects of DADS, such as p21WAF1 expression, seem to be instigated via this mechanism.103,104 Besides DADS, the polysulfanes DATS and DATTS also seem to influence various cellular signaling pathways (possibly by a range of different underlying chemical reactions; see section 3). DATS inhibits the PI3K/Akt pathway and activates the GSK3 pathway.105 Furthermore, DATS leads to an increase in the phosphorylation and thereby activation of the p38 MAP kinase, which is a member of the mitogen activating kinase signaling cascade. Moreover, DATTS influences the DNA damage inducible signaling pathway including chk1 and chk2 kinases, the downstream cdc25 phosphatases, and the downstream cell cycle regulating cyclin B/cdk1 kinase complex.106,107 In addition, various members of the signaling cascade leading to apoptosis, such as Bax, Bcl-2, Bim, Bak, and Bad are affected by DATS or DATTS, although the proteins affected seem to vary between different tumors.101,108−110 Notably, intracellular Ca2+ ion concentrations are also influenced by diallyl polysulfanes.111−114 Not all cysteine modifying agents affect the cell cycle or induce apoptosis. As predicted by the model of Schafer and Buettner, the redox state of the cell may also impact on cell differentiation.99 Recent studies have shown that 1,2-DT can interfere with the formation of adipocytes, which is of importance in the context of obesity and weight control. 1,2-DT affects differentiation and inflammation of human preadipocytes. The underlying (bio)chemical processes are still largely unknown but may involve the

2-fold: On the one hand, the cysteine residues in question sequester oxidants and hence protect the cell from oxidative damage. On the other hand, many of the proteins and enzymes affected lose or change their function and activity, which results in a widespread disruption of the cellular process and influences cellular signaling. Unless OS subsides within a reasonable period of time, such cells become overwhelmed by oxidizing species and ultimately tend to undergo redox-induced apoptosis. These processes are apparently controlled by a variety of redox-sensitive cysteine proteins, including some of the peroxiredoxins (Prdx) and some of the Bcl-2 proteins.93−95 Prdx II, in particular, seems to employ the full range of cysteine chemistry in order to act as an efficient redox-sensor and floodgate for H2O2.96 Under normal conditions and in the presence of mild OS, this enzyme catalyzes the reduction of H2O2 in the presence of thiols. As part of the catalytic cycle, the active site cysteine residue(s) occurs as thiol, sulfenic acid, and disulfide. Under more severe conditions of OS, however, (one of) the active site cysteine residues becomes overoxidized to a sulfinic acid, the antioxidant activity of the enzyme is subsequently switched off (or switched to an activity as a cellular chaperone), and the floodgate for H2O2 is opened up.93,95,97,98 Since H2O2 is no longer removed by Prdx II, it accumulates within the cell, oxidizes various key proteins (perhaps including antiapoptotic Bcl-2 proteins), and subsequently causes cell death. While PrdxII seems to be redox regulated, it is worth pointing out that other Prdx enzymes are primarily controlled by phosphorylation, such as Prdx I.96 In contrast to this scenario of a severe oxidative insult, the cellular response toward milder forms of oxidative stress or indeed to subtle redox processes triggered by selective oxidants is considerably more differentiated. Here, the posttranslational modification of key redox sensors allows the cell to respond appropriately to a change in redox potential, for instance by activating antioxidant systems. As already mentioned, not all cysteine residues in proteins and enzymes react in a similar manner. Some cysteine residues are more reactive, while others are hardly modified. Factors such as the individual pKa or Epa value of a given cysteine residue, its accessibility, and proximity to the modifying agent all affect the reactivity and hence the modifications to be expected. Besides simple reactivity (often described in terms of pKa or Epa values), the location of the proteins to be modified and the position of the cysteine residues within these proteins are clearly of paramount importance.21 In order to understand a wider cellular response, we also need to realize that most cysteine proteins and enzymes theoretically fulfill two functions within the cell which merge at the level of the individual cysteine residue(s). On the one hand, cysteine proteins and enzymes carry out a specific task, such as catalysis, signaling, or regulation (e.g., cdc25 phosphatases and caspases). On the other hand, they are also sensitive toward (oxidative) modifications of the thiol group(s), modifications which interfere with their function and activity, and turn these proteins and enzymes into prime sensors and ef fectors for specific redox signals and more general changes in the cellular redox environment. Oxidation of cysteine residues in Keap-1, for instance, releases Nrf2 and hence enables the cell to mount an antioxidant defense (see section 3), while oxidation of cdc25 enzymes may result in cell cycle arrest and ultimately in apoptosis. Here, the concept of gradual and fairly selective modification of individual cysteine residues in specific proteins based on differences in Epa values and, as the example of Prdx II has illustrated, also via different sulfur oxidation states 599



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given residue is accessible for (oxidative) modification by a modifying agent. Hence, a simple ranking based on Epa values may represent a first step only, which has to be followed by considerably more extensive considerations, probably also in the form of computational approaches. This area of research requires the urgent attention of redox- and electrochemists. At the same time, our knowledge regarding the possible biochemical implications of such cysteine-modifications is still rather limited. The past decade has witnessed the emergence of a number of cellular signaling cascades where cysteine-proteins are involved, including a couple of prominent cysteine-rich proteins which may serve as cysteine-based redox sensors able to trigger or to convey signals related to changes in the intracellular redox state. Keap-1, for instance, contains 27 cysteine residues and appears to be such a regulatory protein.117 Nonetheless, there may be many others. A recent report by Raj et al. has identified glutathione S-transferase pi 1 (GSTP1) as one of the most prominent targets of piperlongumine, a substance able to selectively kill cancer cells by interfering with the intracellular stress response to ROS.118 Indeed, GSTP1, in addition to its conventional function as a detoxifying glutathione S-transferase, may also play an important role in S-glutathiolation of specific target proteins and in the maintenance of the cellular redox homeostasis.119 Not surprisingly, the precise events triggering and subsequently controlling such intracellular regulatory signaling cascades are still not entirely understood. We know, for instance, that diallyl polysulfides cause apoptosis in certain cancer cells; yet we do not understand how such polysulfanes react within the cell (and with which specific target(s)) in order to initiate the apoptotic pathway(s). Do such compounds modify one particular cysteine protein, do they cause a more widespread shift of the GSSG/GSH ratio (and hence an increase in S-thiolation), or do they form ROS and hence indirectly oxidize, randomly, many cysteine proteins? Then again, diallyl polysulfanes may not interfere directly with proteins of the apoptotic pathway at all; they may rather cause serious damage to the cell (e.g., via adventitious interactions with membranes, metalloproteins, or β-tubulin), which would only subsequently trigger apoptosis. At the same time, many of the compounds considered here are based on a reactive, sulfur-containing motif. While such agents may well modify cysteine residues in proteins and enzymes, several of them can also (simultaneously) release H2S, a potent biological control and signaling molecule. Indeed, there are some recent reports describing cysteine-activated H2S donors, which include DATS and the dithiolethione S-diclofenac.120 This inorganic aspect of sulfur chemistry is often ignored, yet may represent a major cause of the biological activity associated with many of these compounds, including dithiolethiones, thiocarbamates, and polysulfanes. Once our knowledge about the cellular targets, when and how they are hit by cysteine modifying agents and which consequences this may have, has improved, it will be possible for chemistry to deliver the compounds able to interfere with these targets in a highly efficient and selective manner. It should be mentioned that cysteine is not the only redox sensitive amino acid which may be affected by changes of the intracellular redox environment. It appears that the oxidation of specific methionine residues in proteins and enzymes may have a similar biochemical impact, despite the fact that methionine residues are seldom catalytically active. Here, oxidation of the methionine sulfide to a sulfoxide or sulfone, as well as (partial)

reduction of PPARγ2 activity and a reduced expression and secretion of leptin and adiponectin.115 If and how these effects are related to the redox activity of 1,2-DT is still unclear. In general, the cellular targets of cysteine modifying agents, i.e., cysteine proteins affected by redox processes, are not easy to identify: Not all cysteine containing proteins and enzymes are per se sensitive to redox modifications, and even if such a modification in a particular protein occurs, its impact on biochemical processes, if any, needs to be confirmed as a part of complicated and time-consuming cellular studies. Furthermore, the biochemical mechanisms underlying the activity of many of these cysteine-modifying agents are also not fully understood. This is hardly surprising since the cysteine modifying agents are chemically diverse, result in a range of distinct cysteine modifications, and, depending on the protein or enzyme affected, may trigger a plethora of different biochemical processes.

6. CONCLUSIONS The previous sections have demonstrated the considerable potential that specific cysteine-modifying agents hold in the field of drug design and pesticide development. Research in this area is fuelled by recent developments in biochemistry and synthetic organic chemistry. In biochemistry, the past decade has witnessed a considerable interest in cysteine-centered redox regulation and sulfur oxidation states and has seen the emergence of the cellular thiolstat as a complex network of redox-controlled cysteine proteins and enzymes. At the same time, synthetic chemistry and biosynthetic chemistry both have provided a range of novel and sometimes rather unusual sulfur compounds with promising chemical and biochemical properties. Some of these agents ultimately may even allow us to selectively target very specific cysteine proteins and enzymes. Nonetheless, a number of obstacles still remain. First of all, we still do not fully understand the processes which govern cysteine modifications in vivo. Which modification occurs when, where, and under which conditions still seems to be highly unpredictable. In some instances, different cysteine residues in the same protein appear to be affected differently. At the same time, often only a fraction of a given protein in the cell is actually modified. Furthermore, not all cysteine residues are equally modified, for instance by S-thiolation; different oxidation products can be found, including intra- and intermolecular disulfides, S-nitrosated residues, sulfenic, and sulfinic acids. Recently, the notion of trisulfide (more properly trisulfane) formation in proteins has gathered momentum, opening up a further and whole new field of thiol and disulfide modifications in proteins and enzymes. The techniques currently available to analyze such modifications inside the cell are often cumbersome, situation-dependent, and still not very reliable.116 Alternatively, a ranking of redox sensitive cysteine proteins may be compiled in vitro first, with the hope of identifying the most sensitive proteins upfront and to subsequently hunt for their modified forms inside the cell. Unfortunately, redox sensitivity is a relative term, often dependent on the oxidant and experimental in vitro conditions employed. Not surprisingly, reliable Epa values or redox potentials would be most useful yet so far have been reported for only a few cysteine proteins and enzymes. At the same time, the matter of location, i.e., the location of proteins within the cell, as well as the location of specific target cysteine residues within proteins, is of utmost importance, as it decides if a particular protein is actually in proximity to a modifying agent (or not) and if a 600



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deacetylase; Isotope-ABPP, isotopic tandem orthogonal proteolysis-activity-based protein profiling; Keap-1, Kelch-like ECH-associated protein 1; NBD-Cl, 7-chloro-4-nitrobenzo-2oxa-1,3-diazole; NEM, N-ethylmaleimide; NHE, normal hydrogen electrode; NOS, nitric oxide synthase; OS, oxidative stress; PDI, protein disulfide isomerase; Pr, protein; Prdx, peroxiredoxin; PTEN, phosphatase tensin homologue on chromosome 10; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species; Srx, sulfiredoxin; Trx, thioredoxin; TrxR, thioredoxin reductase

reversal of this process in the presence of methionine sulfoxide reductase enzymes, provides an interesting lead for future investigations. At the same time, research should also pay more attention to the various enzymes involved in the control of posttranslational cysteine modifications. Within this context, modifying agents of proteins such as the thioredoxins (Trx) and sulfiredoxin (Srx), and inhibitors of enzymes such as the glutaredoxins (Grx), thioredoxin reductase (TrxR), Prdx and PDI, bear considerable potential. Such inhibitors may trigger a fairly selective accumulation of oxidized or S-thiolated proteins inside the cell, which in turn may cause specific and well-defined cellular responses. These inhibitors are joined by a range of new substances, including piperlongumine, which target emerging regulatory enzymes, such as GSTPi.118 Since most of the proteins and enzymes regulating posttranslational cysteine modifications also contain active site cysteine residues, the design of selective inhibitors for these proteins/enzymes is far from trivial. Nonetheless, some of the targets mentioned contain highly reactive cysteine residues or, in the case of GPx and TrxR, even a reactive selenocysteine residue, which stand out and may be hit rather selectively. Indeed, there are already several promising examples of such inhibitors, some of which exhibit considerable activity. Metal-based inhibitors of human TrxR, such as the gold compound auranofin, for instance, possess an extraordinarily high affinity for the selenocysteine residue in this enzyme and are able to trigger apoptosis in certain cancer cells. Similarly, conoidin A is a covalent inhibitor of protozoan Prdx, which prevents host cell invasion by the parasite Toxoplasma gondii.121 Even DADS has been considered as an inhibitor of such enzymes (especially TrxR), although it still remains unclear why such enzymes would be the prime target(s) of this otherwise unspecific disulfide. Overall, posttranslational cysteine modifications in proteins and enzymes represent a wide, diverse, and open field of research which nowadays bears considerable promise for innovative drug design and pesticide development.

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AUTHOR INFORMATION Corresponding Author *Tel: +49 (0)681 302 3129. Fax: +49 (0)681 302 3464. E-mail: [email protected]. Funding We acknowledge the financial support of the University of Saarland, the Ministry of Economics and Science of Saarland, the Deutsche Forschungsgemeinschaft (DFG grants JA1741/2-1), and the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement 215009 RedCat.

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ACKNOWLEDGMENTS We thank Dr. Lalla Aicha Ba, Uma M. Viswanathan, and Aman K. K. Bhasin for helpful discussions, as well as for assistance with literature mining and some of the figures.

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ABBREVIATIONS 4-HNE, 4-hydroxy-2-nonenal; DADS, diallyl disulfide; DAS, diallylsulfide; DATS, diallyl trisulfide; DATTS, diallyl tetrasulfide; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); Epa, anodic (oxidation) potential; Epc, cathodic (reduction) potential; EPR, electron paramagnetic resonance; GPx, glutathione peroxidase; Grx, glutaredoxin; GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione S-transferase; HDAC, histone 601



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Control of Oxidative Posttranslational Cysteine Modifications: From

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