TY - JOUR
AU - Rocha, João B T
AB - Abstract Selenium is an essential trace element for animals and its role in the chemistry of life relies on a unique functional group: the selenol (–SeH) group. The selenol group participates in critical redox reactions. The antioxidant enzymes glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) exemplify important selenoproteins. The selenol group shares several chemical properties with the thiol group (–SH), but it is much more reactive than the sulfur analogue. The substitution of S by Se has been exploited in organic synthesis for a long time, but in the last 4 decades the re-discovery of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) and the demonstration that it has antioxidant and therapeutic properties has renovated interest in the field. The ability of ebselen to mimic the reaction catalyzed by GPx has been viewed as the most important molecular mechanism of action of this class of compound. The term GPx-like or thiol peroxidase-like reaction was previously coined in the field and it is now accepted as the most important chemical attribute of organoselenium compounds. Here, we will critically review the literature on the capacity of organoselenium compounds to mimic selenoproteins (particularly GPx) and discuss some of the bottlenecks in the field. Although the GPx-like activity of organoselenium compounds contributes to their pharmacological effects, the superestimation of the GPx-like activity has to be questioned. The ability of these compounds to oxidize the thiol groups of proteins (the thiol modifier effects of organoselenium compounds) and to spare selenoproteins from inactivation by soft-electrophiles (MeHg+, Hg2+, Cd2+, etc.) might be more relevant for the explanation of their pharmacological effects than their GPx-like activity. In our view, the exploitation of the thiol modifier properties of organoselenium compounds can be harnessed more rationally than the use of low mass molecular structures to mimic the activity of high mass macromolecules that have been shaped by millions to billions of years of evolution. Graphical Abstract Open in new tabDownload slide Here, we critically review the literature on the capacity of organoselenium compounds to mimic selenoproteins (particularly GPx) and discuss some of the bottlenecks in the field. Introduction Selenium is the heaviest of the elements found in a few informational macromolecules (proteins) of different types of organism. In the selenoprotein family, the Se atom is bound covalently to a C atom, creating an organic molecule containing Se. Animals, and particularly vertebrates, have dozens of specific selenoproteins, which contain at least one selenocysteine residue.1–10 The selenocysteinyl residue has a selenol group that is analogous to the thiol group found in cysteine and it is an important redox center in selenoproteins.1–4,10 Selenium can also be found in other amino acids, for instance in selenomethionine; however, in this case, selenium is incorporated in a non-specific way replacing the sulfur atom in plants, particularly, in grains.11,12 In mammals, the ingestion of plant proteins containing selenomethione will result in the incorporation of selenomethione into their proteins in the place of methionine. Although this incorporation is non-specific, it can hypothetically modify the function of proteins and enzymes containing methionyl residues that are in close proximity to biochemically active moieties.11 The differences between the specific (selenium in the selenocysteinyl residues of selenoproteins) and non-specific incorporation of selenium into proteins (e.g., selenomethionine) are depicted in Fig. 1. The incorporation of selenocysteine into selenoproteins is guided by a specific codon (the UGA codon), whereas the non-specific incorporation of selenomethione will occur in sites with the methionine code (AUG). Fig. 1 Open in new tabDownload slide Schematic representation of a selenoprotein (which contains a selenol group) and a protein containing selenium (which contains selenomethionyl residues incorporated non-specifically). The selenocysteinyl residue (Sec) is from GPx-4 (PDB: 2OBI)13 and the selenomethionyl residue (Mse) is from the Interferon gamma receptor (PDB: 4EQ3).14 The chemical and biological interest in selenium dates back to the epoch of selenium’s discovery.10 The similar chemical behavior of tellurium-, selenium- and sulfur-containing compounds has been well known for decades and the substitution of sulfur by selenium or tellurium in organic synthesis goes back to the time of selenium's emergence. However, the interest in the potential pharmacological effects of inorganic and organoselenium compounds commenced decades later. Colloidal selenium (elemental selenium) was the first preparation of selenium used for medical purposes in humans.10,15 According to the physician E. Watson-Williams from Bristol, “the brick-red selenium-β” was the first type of elemental selenium formulation used medicinally for cancer treatment in 1833.15 According to Watson-Williams, various formulations of “powders and pastes” were available on the market and employed by physicians for “the relief of malignant ulcers”.15 The employment of elemental or colloidal selenium in the treatment of inoperable tumors was discontinued some decades later without a clear explanation. The demonstration that selenium was involved in the poisoning of livestock from specific regions containing high levels of selenium in soil, pasture and grain16–18 likely contributed to the abrupt abandonment of elemental selenium in medicine. In fact, the history of selenium has been marked by a duality between its toxic and beneficial effects to mammals.18,19 More recently, the re-emergence of ebselen (2-phenyl-1,2-benzoselenazol-3-one), which was originally synthesized in 1924,10 and the demonstration that it has therapeutic properties has renewed interest in the organic synthesis of new seleno organic compounds.20–42 In fact, ebselen is considered a clinically safe drug for humans and it is included in the National Institutes of Health (NIH) Clinical Collection.32 However, ebselen has not yet been approved for the treatment of any specific diseases. In Japan, ebselen was used in clinical trials for the treatment of brain insults associated with oxidative stress with borderline efficacy.23–25 However, the Japanese government did not approve its clinical use for brain ischemia/stroke.30 Nowadays, ebselen is being tested as a potential lithium mimetic by researchers in the UK31–35 and, according to Nosengo,36 a group from Oxford University has contracted with a pharmaceutical company to carry out clinical trials with ebselen for the possible treatment of bipolar disorders. Ebselen has also entered in clinical trials to treat hearing loss caused by loud music37,38 and vascular problems caused by diabetes.39 More recently, the search for potential new “targets” of ebselen has increased40–44 and new synthetic approaches have been applied to synthesize new ebselen derivatives or to bind ebselen covalently with therapeutic drugs.45–49 The partially successful history of ebselen as a therapeutic agent candidate will not be detailed here, but interested readers may wish to review recent reports on ebselen development for use as a drug.29,30 An aspect to be emphasized here is the role played by the antioxidant properties of the “re-discovered selenium compound” 2-phenyl-1,2-benzisoselenazol-3(2H)-one or ebselen20,21,50–54 in its pharmaceutical exploitation. Indeed, the capacity of ebselen to degrade H2O2 in the presence of reduced glutathione (GSH) or other thiol-containing molecules20,21,31,51 brought ebselen and organoseleno-compounds into the biological scenario. The ability of ebselen to partially mimic the catalytic activity of glutathione peroxidase led to the creation and establishment of the term “glutathione-peroxidase or thiol-peroxidase-like activity” in the chemical and pharmacological field of organochalcogens.20,21,51 The present review aims to clarify how the glutathione-peroxidase-like activity (GPx-like activity) of organoseleno-compounds was crucial in the development of this field of research, particularly the development of new synthetic organoselenium compounds as promising pharmacological agents (for reviews see ref.20,21,55–61). Here we will critically discuss the pros and cons of emphasizing the GPx-like activity as the most important chemical property of organoselenium compounds and whether it may be considered the most important cue to be followed by chemists and pharmacologists in search of novel and even more effective selenium compounds. Physiological roles of selenium in living cells The toxicity of inorganic selenium salts to microorganisms and mammals was characterized some years after the isolation of the element by Berzelius.10 However, the first demonstration that selenium was essential to Escherichia coli was made in 1954 by Jane Pinsent.62 She demonstrated that selenite was required in the synthesis of active methanoate dehydrogenase. In 1957, Swartz63 and collaborators reported that selenium could prevent hepatic necrosis induced by a diet deficient in vitamin E. However, the study of Swartz and Foltz did not establish a physiological, or biochemical role for selenium in mammals. Subsequently, indirect evidence that selenium (selenite) has antioxidant properties was obtained by Tappel and co-workers using mammalian and avian models of vitamin E deficiency.64,65 These researchers demonstrated that addition of selenite (Se4+) to basal diets or diets deficient in vitamin E decreased the spontaneous lipid peroxidation in different tissues, such as liver, kidneys and muscle.64–66 However, the demonstration that selenium participates in a specific molecular biochemical reaction was made only in 1973 by two independent groups.67,68 The European and American groups demonstrated that selenium was tightly associated with purified bovine or rat blood glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, E.C. 1.11.1.9). The characterization of erythrocyte glutathione peroxidase, an antioxidant enzyme described earlier by Mills69–71 as a selenoenzyme, supported the observations made by Tappel and collaborators that selenium had antioxidant roles in vertebrates.64–66 Cytosolic glutathione peroxidase (GPx1) catalyzes the degradation of hydrogen peroxide (H2O2).19,72,73 The basic reaction catalyzed by GPx is: 2GSH+H2O2→GSSG+2H2O Active GPx1 is an homotetrameric enzyme with a molecular weight of 84 kD and 1 Se atom/monomer.67,74 The enzyme was crystallized in 197675,76 and the presence of selenium as a selenocysteinyl residue (Fig. 1 left panel and Fig. 2) was confirmed in hepatic rat glutathione peroxidase.77 The presence of selenocysteinyl in the active center of the bacterial glycine reductase was confirmed two years before.78 Fig. 2 Open in new tabDownload slide Glutathione peroxidase structure of GPx-1, -2 and -4: (A) the structure of the tetrameric native GPx1 (active GPx2 is also a tetramer, whereas GPX4 is active in the monomeric form); (B) the catalytic triad containing the selenol group of the selenocysteinyl residue interacting with tryptophanyl and glutaminyl lateral groups. The inactive monomer of GPx-1 (C) and GPx-2; the GPx-4 active monomer (E). The distances between the selenium atom and the nitrogen atoms varies from 3.2 to 3.9 A. The catalytic triad is also depicted in the monomers of GPx-1 (C), GPx-2 (D) and GPx-4 (E). The similarity between the triads is evident. The structures are based on the X-ray analysis of GPx-1 performed by Epp et al.76 (PDB ID: 1GP1), GPx-2 performed by Johansson et al.84 (PDB ID: 2HE3) and GPx-4 by Scheerer et al.13 (PDB ID: 2OBI). The cysteinyl S atoms were replaced with Se to regenerate the original selenoenzymes. The structures were analyzed using the program Accelrys Discovery Studio 3.5.85 Studies on the mechanism of the catalysis of GPx were pioneered by Prof. Flohé and collaborators, who worked on the enzyme even before there was any knowledge that it was a selenoenzyme.79–82 The most plausible catalytic cycle of GPx is depicted in Fig. 3.76,81–83 In this cycle, the selenol group of GPx plays a fundamental role in the degradation of peroxides.76,77 As depicted in Fig. 3, the selenolate (selenol) group of the enzyme can attack the peroxides, reducing them to H2O or to an alcohol. The glutathione peroxidase isoforms have a common catalytic triad formed by the selenocysteinyl, glutaminyl and tryptophanyl residues (Fig. 2).76 The proximity of the glutaminyl and tryptophanyl residues to the selenocysteinyl residue facilitates the dissociation of the selenol to the selenolate state. As a result, the selenocysteinyl residue is rapidly oxidized by hydroperoxides to a selenenic acid intermediate. Subsequently, selenenic acid is attacked by a thiolate of a reduced glutathione to form the intermediate GSSeGPx. The intermediate containing the –S–Se– bond is further reduced by a second GSH, releasing the oxidized glutathione (GS-SG) and the active GPx. Fig. 3 Open in new tabDownload slide The catalytic cycle of the seleno-glutathione peroxidase isoforms. The selenol/selenolate functional group reacts with peroxide, forming a selenenic acid intermediate of the enzyme. Subsequently, the oxidized atom of selenium reacts, in two sequential steps, with GSH, regenerating the active enzyme. In subsequent years, the search for new selenoproteins revealed that 75Se-labeled selenite was incorporated into various selenoproteins (selenocysteine residues of about 7 to 9 selenoproteins) and a greater proportion of 75Se was incorporated into classical GPx.86–88 In this way, Ursini and collaborators demonstrated that the “peroxidation-inhibiting protein” purified from the tissues of rodents89 was a glutathione peroxidase.90 The purified selenoenzyme had a monomeric structure of about 23 kDa and efficiently reduced the membrane bound hydroperoxides and phospholipid hydroperoxides, using GSH as a reducing equivalent donor.90 Phospholipid hydroperoxide glutathione peroxidase is now classified as GPx4 and three main isoforms of GPx4 have been described: the cytosolic (cGPx4), mitochondrial (mGPx4) and sperm nuclear (snGPx4) isoforms.91,92 As depicted in Fig. 2, the structure of the active site of phospholipid hydroperoxide glutathione peroxidase (GPx4)13 is similar to the inactive monomer of GPx1. However, GPx4 lacks an internal sequence of 20 amino acids in a surface loop that regulates substrate specificity in GPx1 and other GPx enzymes.93–97 To date, a total of 8 forms of GPx enzymes have been found in the human genome, of which 5 are selenoenzymes (GPx1-GPx4 and GPx6), while the others are thiol enzymes. GPx2 is structurally similar to GPx1 and is expressed mainly in the gastrointestinal tract. GPx3 is also structurally related to GPx1 and is an extracellular enzyme that can be found in plasma. More recently, the literature has indicated that GPx can bind to the basement membrane in the kidney, epididymis and bronchi, among others. All the 5 selenoenzymes are involved in the maintenance of peroxide levels and can control different signaling cascades. For instance, GPx4 has recently been shown to play a central role in different types of cell death, for instance, apoptosis and ferroptosis.73,98–102 As GPx4 is a unique antioxidant enzyme that directly reduces peroxidized phospholipids generated in the cell membrane, it has been shown to be a key regulator of a novel oxidative and iron dependent form of regulated cell death called ferroptosis. Ferroptosis is characterized by the accumulation of lipid peroxidation products and high production of iron-dependent ROS due to the GSH/GPx4 system dysfunctions.103–107 In effect, the inhibition of cystine-glutamate antiporter xc− that transports cystine into cells for GSH synthesis and inactivation and/or down-expression of GPx4 are considered triggers of ferroptosis (Fig. 4). Fig. 4 Open in new tabDownload slide Ferroptosis induction and organoselenium compounds as GPx4 mimetics. (A) Glutathione (GSH) is used as a substrate by GPx4 to prevent lipid hydroperoxide accumulation. The GSH is synthesized from cystine (Cys2), which is imported by system xc−. (B) GPx4 system inhibition leads to lipid hydroperoxide accumulation and causes ferroptosis. (C) Organoselenium compounds can act as a GPx4 mimetic, preventing lipid hydroperoxide accumulation and consequently ferroptosis. PUFA: Polyunsaturated fatty acids. In addition to iron-chelating compounds, ferroptosis is significantly inhibited by antioxidants (i.e., α-tocopherol, β-carotene, linoleic acid, butylated hydroxytoluene, ciclopiroxolamine and trolox).103–107 Specifically toward GPx mimics, Dixon and collaborators demonstrated the efficacy of ebselen in blocking the death induced by erastin and RSL3 in cancer cells, molecules respectively known by directly inhibiting the system xc− and GPx4 activity.103 The implication of ferroptosis in several pathophysiological processes strengthens the therapeutic interest in targeting organoselenium compounds that could act to suppress the death at the level of GPx4 mimicry or lipid peroxidation inhibition. In conformity with this, an elegant study recently published argued that due to the essentiality for GPx4 expression and activity, adequate selenium levels could protect the brain against the neurodegeneration caused by ferroptosis.99 GPx4 has different physiological functions related to the reduction of lipid hydroperoxides, including the regulation of lipoxygenase and cyclooxygenase (consequently, GPx4 can modulate inflammatory response).92 The description of selenocysteinyl residues in the selenoenzymes of prokaryotes and eukaryotes shows the universality of selenium in living organisms.5,6 Indeed, to date, the unique functional organic group of selenium in living cells seems to be the selenol group found in the selenocysteinyl residues of selenoproteins. However, selenoproteins were apparently lost by several groups of living organism, for instance, higher plants and fungi do not have selenoproteins.6 The majority of selenoproteins participate in important redox reactions and in mammals a good portion of the selenoproteins, if not all, are antioxidant enzymes essential for cell physiology and homeostasis (for recent reviews see ref. 1–3 and 8–10). So far, 25 selenoproteins have been identified in the human genome by means of bioinformatic and biochemical tools.3,4 However, the possibility of finding new selenoproteins still exists. Glutathione peroxidase, thioredoxin reductase and iodothyronine deiodinase isoenzymes are the three main studied classes of mammalian selenoprotein.1–3,7–10 As noted above, GPx was the first identified selenoprotein in mammals. The identification of selenocysteinyl residues in some proteins brought to the biological scenario a very reactive and the softest nucleophilic center of biomolecules, i.e., the selenol group. Indeed, the selenol group is an important redox center in living cells.108 Low molecular mass organoselenium As noted in the previous section, selenocysteine does not exist as a free amino acid, because its selenol group is highly reactive. The synthesis of selenocysteine occurs co-translationally at the level of selenocysteine t-RNA (Sec-Ser-t-RNA) and it is tightly regulated to fulfill the physiological demand for selenoprotein synthesis. Thus, the release of a negligible quantity of selenocysteine will occur only during the degradation of selenoproteins. However, recent studies have indicated beta-cleavage of the oxidized selenocysteinyl residue, releasing methylselenol from denatured GPx isoforms and from selenoprotein-P.109–111 When the latter occurs during the normal turnover of all selenoproteins, the release of free selonocysteine may be non-existent or undetectable in vivo. However, it is possible that noticeable levels of free Secys may be found in circulation and other fluids (e.g. milk) as a corollary of the ingestion of vegetables and animal products containing significant amounts of selenium species as Secys, SeMet and Se-methyl-selenocysteine.112 However, in aerobic and aqueous media of living cells, any hypothetical excess of synthesized selenocysteine will be rapidly oxidized to selenocystine or can even be decomposed as observed in some selenoproteins.109–113 In fact, living cells do not have appreciable amounts of low mass organoselenium compounds with free selenol groups. Though, selenometabolites of low molecular mass have been detected in human plasma/serum samples and cow milk.112,114 The authors have not demonstrated specific compounds with free selenol groups. In accordance, low molecular mass selenium compounds (selenosugars and methyl-selenoneine) are found in human urine.115 In contrast, they did not report the presence of selenoneine (2-selenyl-Nα, Nα, Nα-trimethyl-l-histidine; which has a selenol group) in their samples. The presence of selenoneine has been reported in fish,116–120 but the compound was reported to be very unstable and easily oxidized to its diselenide form. Low molecular mass organoselenium compounds can also be found at appreciable quantities in plants as a result of the non-specific incorporation of selenium in the place of sulfur.11,121,122 In fact, plants adapted to live in selenium rich soil can accumulate specific low molecular mass selenium-containing compounds, such as methyl-selenocysteine.122 In this type of plant, the non-specifically synthesized selenocysteine has its reactive selenol group methylated, possibly to avoid the toxicity of high concentrations of free selenol. As a corollary, we posit that low molecular mass compounds containing selenol groups are not physiologically exploited by living cells and that organisms that synthesize large amounts of selenol-containing molecules have mechanisms to metabolize it to non-reactive intermediates (e.g. methyl-selenocysteine). However, the interest in naturally occurring organoselenium compounds increased considerably after the implication of the presence of selenium in plants in the poisoning of farm livestock in the USA.10,16–18 Here, we will not discuss the toxicology of selenium and the reader may consult several comprehensive reviews and references herein.10,16,17,21 From a chemical perspective, the exploitation of selenium in organic synthesis started soon after the isolation of the element by Berzelius and this persists to date.10,123 The versatility of selenium in organic synthesis as a substitute for sulfur or tellurium was well-known by earlier chemists, for instance the renowned Friedrich Wöhler. At the end of the 19th century the number of organoselenium compounds synthesized in the laboratory was limited by the availability of the element to chemists (for more details on the history of Se use in organic synthesis see ref. 10). Here we will not discuss the earlier use of organoselenium compounds as potential pharmacological agents and the reader may refer to classical or more recent reviews.10,16,21 Briefly, we will discuss how the apparent serendipity of the re-discovery of ebselen changed the course of the organic synthesis of low molecular mass selenium-containing compounds. Ebselen was first synthesized in 1924 and in the 1970's it was rediscovered as a potential antioxidant and therapeutic agent.10,28,29 Here we focus our review on a few classes of low molecular mass organoselenium compound that can partially mimic the physiological activity of some selenoproteins, particularly the GPx isoforms. Selenium compounds that can mimic selenoproteins The reaction catalyzed by GPx can be imitated by several selenium containing molecules. However, the mechanism of “mimicry” of GPx by organoselenium compounds can vary depending on the class of compound considered. Fig. 5 depicts several prototypical molecules that can mimic the GPx reaction. Basically, we will refer here to diorganyl selenide (or monoselenides) and diorganyl diselenide (or diselenides). Important molecules of these two classes are the well-known ebselen, ebselen diselenide and other diselenides that can be transformed into selenol intermediates. The selenol intermediates can catalyze the decomposition of peroxides (both inorganic and organic peroxides) via reactions that, to a certain extent, mimic the native isoforms of GPx. Fig. 5 Open in new tabDownload slide General representation of classes of low-molecular mass organoselenium compound that can mimic the selenoenzyme GPx. The molecules presented here include ebselen (which is, from a biological point of view, the most studied organoselenium compound), diselenides, selenophenes, peridiselenides and spirodioxaselenuranes. The main pathway of peroxide degradation by ebselen and diselenides involves the formation of selenol intermediates (in the case of the diselenide containing the nicotinoyl based aromatic moiety, the selenol (–C–SeH) is in equilibrium with the selone form (–C˭Se), which is the active intermediate in the decomposition of peroxides). In the first three reactions, the formation of a selenenic acid intermediate after the oxidation of the selenium atom by peroxides is analogous to the intermediate formed in the native GPx seleno-isoforms (Fig. 3). In the case of reactions (4)–(6), the intermediates formed are selenoxides and their peroxide decomposition pathways are not equal to that of the native enzyme. In a broad sense, we posit that ebselen and diselenides can be seen as “hidden selenol-containing molecules” (Fig. 5). The formation of the selenol intermediates can occur either by a direct reaction of the parent compounds with thiol-containing molecules, e.g., reduced glutathione (GSH) or thiol-containing proteins, or by the action of the selenoenzyme thioredoxin reductase (TrxR), which catalyzes the transfer of reducing equivalents from NADPH to ebselen, ebselen diselenide, diphenyl diselenide and other inorganic and organic compounds containing selenium.124–131 In contrast with ebselen (which is a diorganoyl selenide that can be metabolized to the selenol intermediate), other diorganoyl selenide molecules, for instance selenophene, can reduce peroxides by oxidizing the selenium (selenide) to selenoxide intermediates (Fig. 5). The selenoxide can be reduced back to the parent compound by thiol molecules. Ebselen can also be oxidized to its selenoxide intermediate and can be regenerated by thiol reduction. Although the nucleophilicity of selenol/selenolate intermediates is anticipated to be much higher than that of selenide intermediates, there is no systematic experimental comparison between the thiol-peroxidase of these different classes of selenium containing molecule. However, in silico studies have confirmed that the direct oxidation of the selenide to selenoxide in the ebselen molecule is not favored thermodynamically.132 Consequently, the thiol-reduction of the selenium atom in ebselen to the selenenyl-sulfide and selenol intermediates can be considered much more important in the thiol-peroxidase-like activity than the direct interaction of ebselen with oxidizing molecules such as peroxides. Organoselenium compound mimetics of GPx: ebselen Almost three decades have passed since Muller and Wendel and their collaborators22,51 demonstrated that the organoselenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; PZ-51, DR-3305) mimics the activity of GPx in vitro. Since then, a number of synthetic organoselenium containing molecules have been developed as mimics of GPx. Consequently, numerous articles133–138 have described different organoselenium compounds that mimic the native enzyme. As briefly noted above, based on the chemical structure, the synthetic GPx-like compounds can be classified into two major classes: diorganyl selenides and diorganyl diselenides. Diorganyl selenides Cyclic diorganyl selenides The modern biological history of ebselen 1 (Chart 1), the most studied cyclic diorganyl selenide, began in the 1970s. Regarding this intriguing chapter of the history of organoselenium compounds, Parnham and Sies30 retraced the early development of ebselen from their perspective. The pharmacological properties of ebselen26,139–142 have been widely studied during the last three decades,143–150 and advances in the interaction between industry and academia led to novel applications151,152 and repurposing of patents for this organoselenium compound.32–37 Chart 1 Open in new tabDownload slide The observation that ebselen mimics GPx activity was followed by a number of studies on the mechanism for the thiol-dependent catalytic reduction of hydroperoxides by this compound.153–156 In addition to ebselen selenol,157 ebselen selenosulfide and ebselen selenenic acid, other ebselen metabolites (selenoxide and diselenide), with varying oxidation number of the selenium, are thought to be intermediates in the reaction of ebselen (Fig. 6).140 Furthermore, a revised mechanism for the GPx-like activity of ebselen, in which the selenenic acid was the only stable and isolable product after the reaction of ebselen and peroxides, was proposed by Sarma and Mugesh.158 Fig. 6 Open in new tabDownload slide The thiol-peroxidase-like cycle of ebselen. The “mimicry” of the GPx reaction can involve the direct reduction of ebselen by different thiol groups or the indirect reaction catalyzed by TrxR, where the NADPH is used as the source of reducing equivalents for the formation of the selenol/selenolate intermediate. The opening of the –Se–N– bond by thiols results in the formation of an ebselen selenol intermediate that will react with peroxides and thiols in a way analogous to the GPx cycle (Fig. 3). Ebselen and its derivatives have been reported to exhibit moderate catalytic activity, which is attributed to the undesired thiol exchange reactions that take place at the selenium center in the selenenyl sulfide intermediate.159 In this context, Bhabak and Mugesh demonstrated that compounds that produce selenenyl sulfides with strong Se⋯O interactions are less active compared to those with weaker Se⋯O interactions. These authors also demonstrated that the nature of the thiols had a drastic effect on the catalytic activity of ebselen and its analogues.49 Moreover, the introduction of an ortho substituent in an ebselen derivative increased the GPx-like activity, indicating that steric hindrance prevents thiol exchange reactions.160 Although the GPx-like catalytic activity of ebselen is only moderate, and its very low aqueous solubility may be considered a problem in drug administration, the selenium contained in ebselen is not bioavailable for incorporation into the native enzyme GPx.22,50,51 This is considered as a basis for the lack of toxicity observed in experimental studies and clinical trials.23–25,161,162 As ebselen is one of the most promising synthetic antioxidants that exhibits numerous pharmacological activities, this molecule has been used as a model for the synthesis of novel ebselen derivatives163 mimetic of GPx with improved catalytic activity, enhanced water solubility and ease of synthesis. The GPx-like activity of oxygen-substituted compounds49 motivated the synthesis of ebselenols substituted with hydroxy groups in the benzisoselenazolone ring of ebselen. Ebselenols 2, 3, and 4 showed a GPx-like activity similar to that of ebselen, whereas compound 5 was 15-fold more active than ebselen 1 (Chart 1).164 The high activity of 5 was attributed to the proximity effect of the OH group and Se center as previously reported (Chart 2).165,166 Chart 2 Open in new tabDownload slide Considering that ebselen is an acetylcholinesterase (AChE) inhibitor167 and that the impaired function of the cholinergic system has been associated with cognitive deficits, as observed in Alzheimer's disease,168 researchers have pursued the design and synthesis of ebselen derivatives that inhibit the activity of acetylcholinesterase and are good GPx mimetics. Therapeutic compounds for Alzheimer's disease were synthesized by the fusion of ebselen and donepezil, a potent acetylcholinesterase inhibitor. Among the synthesized ebselen derivatives, the inhibitors of acetylcholinesterase activity 6, 745 and 846 as well as the inhibitor of metal-induced Aβ aggregation 9169 (Chart 3) had higher GPx-like activity than that of the parent compound, ebselen 1 (Chart 1). Chart 3 Open in new tabDownload slide The synthesis and GPx-like activity of enlarged ebselen-like ring compounds has also been pursued.170–172 Here, six-membered homologues of ebselen were synthesized and compound 10 (Chart 4) exhibited superior catalytic reduction of hydroperoxide when compared to ebselen 1 (Chart 1).173 The presence of a nitro or methyl group ortho to the selenium atom increased the catalytic activity of ebselen derivatives 11174 and 12175 (Chart 4). Chart 4 Open in new tabDownload slide By contrast, amino acid-containing ebselen analogues with intramolecular coordination had poor catalytic activity at low GSH concentrations, where ebselen 1 (Chart 1) was active.176 The GPx-like activity of di- and tripeptide-based ebselen analogues and the formation of selenol depended on the nature of the peptide moiety attached to the nitrogen atom of the selenazole ring. Therefore, the introduction of aliphatic amino acid residues, but not aromatic, significantly enhanced the GPx-like activity.177 Furthermore, Bhabak and collaborators demonstrated that the incorporation of a suitable electron donating group in the phenyl ring of an aromatic thiol increased the GPx-like activity of amide and amine-based compounds.178 A series of aryl and alkyl ebselen derivatives were synthesized and the effectiveness to mimic the GPx enzyme was reported. The alkyl derivatives with one to four carbon chains exhibited higher reactivity than ebselen. Among the aryl analogues 13–16 (Chart 5), those with the p-nitro, p-iodo and p-methoxy phenyl groups were stronger catalysts than the model compound, ebselen 1 (Chart 1).179 Chart 5 Open in new tabDownload slide In an attempt to develop novel ebselen derivatives with higher thiol-peroxidase-like activity, ebselen chiral analogues were synthesized. The chiral substituent on the nitrogen atom did not provide a substantial increase in the GPx-like activity and the newly synthesized compounds showed analogous activities to ebselen 1 (Chart 1).47 In addition to the studies with ebselen derivatives, several research groups have pursued the design and synthesis of other cyclic diorganyl selenide mimics of GPx, such as selenenamide and related derivatives,180–182 seleninate ester and the related spirodioxaselenanonane,183–186 selenopyrazole,187 selenane188 and selenazolinium salts.189 Singh and collaborators have reported that the seleninate esters exhibited much higher activity than selenenate esters190 and that the seleninamides 17 and 18 are even better catalysts than 19–22.191 In an extension of their work, this research group demonstrated good catalytic activity for selenenate esters 23–25 (Chart 6).192 Chart 6 Open in new tabDownload slide A series of stable spirodiazaselenuranes that catalytically reduce hydroperoxides in the presence of thiols were described. The structure–activity relationship was demonstrated in this study; the introduction of electron donating groups, such as m-OH, significantly enhanced the GPx-like activity of both diaryl selenide 26 and spirodiazaselenurane 27, whereas the introduction of electron withdrawing groups to the phenyl ring decreased the thiol peroxidase-like activity (Chart 7).193 Chart 7 Open in new tabDownload slide A single p-methoxy group substituted in each aryl moiety of diaryl selenide 28 afforded the highest GPx-like activity, while methoxy groups in the meta-position, 29, had little effect compared to the unsubstituted selenide, and o-methoxy groups, 30, suppressed activity. Regarding the introduction of an additional methoxy substituent in the ortho or para position (31 and 32), the pattern in the mimetic catalytic effect is analogous to the one observed with a single methoxy substitution. The trimethoxyaryl derivative 33 was less active than the p-methoxy derivative 28 (Chart 8).194 Chart 8 Open in new tabDownload slide Analogous to the absence of thiol-peroxidase-like activity for aromatic spirocyclic derivatives reported by Tripathi and collaborators195 and Back and collaborators,185 the spirocyclic derivatives 34 and 35, in which the oxidation state of Se is +4, failed to show thiol peroxidase-like activity (Chart 9).196 Chart 9 Open in new tabDownload slide The seven-membered seleninate ester 37 was obtained by the direct oxidation of diselenide 36 with tert-butyl hydroperoxide. This compound showed good GPx mimetic activity.197 The same strategy adopted by Tripathi and coworkers was used to obtain the hydroxymethyl substituted cyclic seleninate 38. Compound 38, synthesized by oxidation of the diselenide 39 or allyl selenide 40 with hydrogen peroxide, showed two fold higher catalytic activity compared to the unsubstituted analogue 41 (Chart 10).198 Chart 10 Open in new tabDownload slide Motivated by the positive effect of the p-methoxy substituent in 42 on the GPx-like activity, a number of cyclic seleninate esters 43–47 substituted with a methoxy group were synthesized and their GPx-like catalytic activity was determined. This study revealed that neither the introduction of single methoxy groups at the ortho or meta position, relative to the selenium center, nor their introduction in addition to an existing p-methoxy group increased the GPx-like activity. While the substitution in 42199 was the most effective, the presence of the o-methoxy group in compounds 44 and 46 hampered the thiol peroxidase-like activity.200 In this context, Bayse and Shoaf demonstrated that the activation barrier for the oxidation of the selenenyl sulfide, a proposed key intermediate in the catalytic cycle, is higher for the o-methoxy derivative than for other positions, consistent with the low experimental conversion rate (Chart 11).201 Chart 11 Open in new tabDownload slide In efforts to increase the bioavailability of cyclic selenides, various lipophilic GPx mimics have been synthesized202 by combining a hydrophilic selenide moiety as a redox center with a long alkyl chain as a lipophilic unit. As a result, lipophilic compounds 48–52 were found to be more potent GPx-like antioxidants than 1 (Chart 1), 53 and 54 (Chart 12).203 Chart 12 Open in new tabDownload slide As the catalytic function of the heterocycles is strongly influenced by the nucleophilic character of the selenium atom and by the presence of free amine groups, benzo[b][1,4]selenazines were prepared and their effectiveness as GPx mimetics was investigated. N-Tosylate derivative 55 did not show catalytic activity, and the corresponding N-unsubstituted derivative 56 (Chart 13) had increased GPx-like activity but was less potent than diphenyl diselenide 53 (Chart 12).204 Chart 13 Open in new tabDownload slide Acyclic diorganyl selenides Research groups working with acyclic diorganyl selenides have synthesized a number of molecules with GPx-like activity, among them α-(phenylselenyl)ketones,205 selenyl naphthol,206 selenyl pyridines,207 selenyl amides,208 diethyl selenides,209 selenocyanate210 and asymmetric alkyl selenides.185 The mechanism for the catalytic cycle of selenide proceeds via a spirodioxaselenanonane intermediate, which is distinct from that employed by the native enzyme and small-molecule mimetics (Fig. 7).211 Fig. 7 Open in new tabDownload slide The thiol-peroxidase-like catalytic cycle of spirodioxaselenurane compounds. The spirodioxaselenurane active compound (in the bottom of the figure) reacts with two thiol reducing equivalents to form an intermediate containing the –Se–S– bond (which is equivalent to the selenylsulfide intermediates found in the native enzyme and in the catalytic cycle of ebselen and diselenides). The intermediate releases the disulfide and the intermediate containing the active reducing selenium compound (top). This compound decomposes the hydrogen peroxide, forming the selenoxide intermediate that spontaneously re-arranges, releasing water and forming the active spirodioxaselenurane compound. Motivated by the catalytic cycle proposed by Back and coworkers, novel GPx mimics of the spirodioxaselenurane type were synthesized. The selenium-containing spiro compounds 58, 59, 60 and 61 (Chart 14) exhibited higher thiol peroxidase-like activity than ebselen 1 (Chart 1).212 Chart 14 Open in new tabDownload slide Water soluble compounds derived from 3-hydroxypropyl or 2,3-dihydroxypropyl parent selenides were highly active catalysts for the reduction of hydrogen peroxide with thiols. In both series the single p-methoxy substituents in 62 and 63 as well as the meta and para disubstituted analogue 64 provided the fastest reaction rates compared to their unsubstituted analogue, 65. This effect was attributed to resonance stabilization of the positive charge on the selenium atom during the rate determining oxidation step (Chart 15).213 Chart 15 Open in new tabDownload slide Compounds 66 and 67 (Chart 16) had higher thiol-peroxidase-like activity than the synthesized derivative 2-(3,5-dimethylpyrazol-1-yl)ethylseleno. The high catalytic activity could be explained by the presence of an amino group which either acts as a good base catalyst for the thiol-peroxidase-like reaction or the basic-like environment produced by the amino groups in the reaction solution facilitating the conversion of reduced dithiothreitol to its oxidized form.214 In an extension of these studies, the symmetrical pyridyl 68 and pyrimidyl monoselenides 69 were reported to have greater GPx-like catalytic activity than that of their unsymmetrical monoselenides, whereas the phenyl mono- and diselenide analogues exhibited poor activity. The electron density around the selenium atom was an important factor influencing the catalytic activity of these compounds.215 The presence of an amino functional group that increases the electron density at the selenium atom was also attributed to the higher catalytic activity of 2-phenyl(3,5-dimethylpyrazol-1-yl)selenides 70 and 71 (Chart 16).216 Chart 16 Open in new tabDownload slide Studies on different substituents in the aromatic ring of beta-selenium amines revealed that the compound 72, which contains a chlorine substituent in the aromatic ring bonded to the selenium atom, exhibited insignificant GPx-like activity. The GPx-like activity of compound 73, without a substituent in the aromatic ring bonded to the selenium atom, was comparable to that of diphenyl diselenide 53 (Chart 12). Compound 74, with a methoxy substituent in the aromatic ring bonded to the selenium atom, had better GPx mimetic activity than diphenyl diselenide 53. Beta-selenium amines 75 and 76, both containing a blocking group bonded to the amino group, did not have GPx-like activity, which suggests the importance of the free amino group in the catalytic activity of beta-selenoamines (Chart 17).218 Chart 17 Open in new tabDownload slide The air stable selenolate 77 was screened toward different thiols in the presence of air or peroxides as stoichiometric oxidants. The compound 77 (Chart 18) promoted the oxidation of thiols reducing hydrogen peroxide via the formation of a selenoxide intermediate.219 Chart 18 Open in new tabDownload slide Selenium-containing chrysin 78 and 3,7,3′,4′-tetramethylquercetin 79, but not their thiol analogues,220 had GPx-like activity higher than selenocarbonyl-containing derivatives, a naphthyl selenourea and a sugar-derived selenourea (Chart 18).221 Regarding the catalytic cycle of diorganyl selenides I, in contrast to diorganyl diselenides, they do not consume thiols for their activation, and are first oxidized to selenoxides II, in the presence of peroxides. In the revised version of the GPx-like catalytic cycle of selenides and selenoxides, Nascimento and coworkers demonstrated that organoselenides do not follow a Se(ii)/Se(iv) redox cycle. In the presence of an excess of hydroperoxide (Scheme 1b), a condition of the GPx-like assay, selenoxides are converted to hydroxy perhydroxy selenanes IV (Scheme 1), which are better oxidizing agents than selenoxides.217 However, under physiological or even at pathological concentrations of hydrogen peroxide, the steps leading to III and IV are unlikely to occur.217 Scheme 1 Open in new tabDownload slide The revised version of the GPx-like catalytic cycle of selenides and selenoxides, adapted from Nascimento et al. (2012).217 Diorganyl diselenides After the discovery of ebselen 1 (Chart 1) as a GPx mimic; several other organoselenium compounds have been reported with chemical properties. Diselenides became quite attractive given their relative ease of synthesis and stability, and, consequently, their suitability for biological investigations.21,222–224 In this section, we will highlight some diselenide derivatives reported in the literature with appreciable GPx-like activity. In comparison with ebselen 1, the GPx- or thiol-peroxidase-like activity of diselenides is commonly higher or analogous to that of ebselen. For instance, diphenyl diselenide 53 (Chart 12), a simple molecule which is known for its efficacy as a GPx mimic and numerous pharmacological properties, has about two fold greater thiol peroxidase-like activity than ebselen.224,225 Diselenides enter the GPx cycle via a reduction mechanism mediated by thiol containing molecules, for instance GSH. This leads to the formation of a selenol intermediate that subsequently reduces the hydroperoxide with concomitant formation of selenenic acid. The oxidized selenium group is regenerated back to selenol by GSH (Fig. 8). Fig. 8 Open in new tabDownload slide The thiol-peroxidase-like cycle of diphenyl diselenide. The “mimicry” of the GPx reaction by various diselenides involves either the direct reduction of the –Se–Se– bond by different thiol groups or the indirect reaction catalyzed by TrxR, where NADPH is used as the source of reducing equivalents for the formation of the selenol/selenolate intermediate. The selenolate reacts with peroxides, forming the selenenic acid that is subsequently reduced by thiols to regenerate the active selenol/selenolate intermediate. One of the first mechanistic studies on the catalytic GPx-like cycle of a diselenide as an enzyme model was conducted by Iwaoka and Tomoda in the 90s.226 Using 77Se NMR spectroscopy, they characterized selenenyl sulfide, selenolate, and selenenic acid as key intermediates in the reaction catalyzed by compound 80 (Chart 19). The role of nitrogen base in the GPx-like activity of the diselenide was related to the following aspects: (1) activation of the selenol intermediate (RSeH) into the kinetically much more reactive selenolate anion (RSe−), (2) the Se–N interaction in the selenenic acid intermediate (RSeOH) preventing its further oxidation into other oxidized selenium species and (3) the Se–N interaction in the selenenyl sulfide intermediate (RSeSG), precluding the nucleophilic attack of the reducing thiol at the Se atom. The attack at the S atom in the selenyl sulfide intermediate allowed the effective regeneration of the selenol intermediate. Chart 19 Open in new tabDownload slide With respect to the catalytic cycle, Mugesh and collaborators investigated the mechanistic properties of several diaryl diselenides having intramolecularly coordinating amino groups.227 The GPx-like activity of the diselenides depended on the strength of the Se⋯N non-bonded interactions in the intermediates produced during the catalytic process. The diselenides having weak Se⋯N interactions, 81 and 82 (Chart 20), exhibited higher activity than the diselenides containing strong Se⋯N interactions, 83 and 84 (Chart 20), due to the fast reaction of their selenenyl sulfide derivatives with thiol to produce diphenyl disulfide and the expected selenol. Chart 20 Open in new tabDownload slide Focusing on amine based diselenides, Wilson and coworkers synthesized and examined the GPx-like activity of a series of tertiary amine analogues of diphenyl diselenide 53.228 The introduction of tertiary amine substituents into the aromatic nucleus at the ortho position to the Se atom in 85 and 86 (Chart 21) increased ≅5-fold the GPx-like activity compared to that of diphenyl diselenide 53 and ≅10 times greater than ebselen 1. The authors assumed that tertiary amines may serve to deprotonate the thiol sulfhydryl group, providing a high local concentration of nucleophilic thiolate anions in the reaction catalysis. Moreover, the conjugate acid of the amine, the ammonium ion, could be serving as a proton source, facilitating the reduction of hydrogen peroxide to water. These assumptions were further supported by the results of a computational study of the reaction profile of compound 85 with hydrogen peroxide (Chart 21).229 Chart 21 Open in new tabDownload slide With respect to the GPx-like activity of diaryl diselenides with tertiary amino group substituents, Bhabak and Mugesh demonstrated that the substitution of the hydrogen atom at the 6-position in N,N-dialkylbenzylamine-based diselenides by a methoxy group markedly increased the catalytic activity.230 The resultant diselenide 87 (Chart 22) exhibited much higher GPx-like activity than ebselen 1. According to the authors, the replacement of an aryl proton in the compound by a methoxy group prevented the Se⋯N interactions in the key intermediates and consequently increased the GPx-like activity. In addition, the incorporation of the electron donating methoxy group in diselenides increases the basic environment of the amino groups, optimizing the Se–N interaction and the catalysis. Chart 22 Open in new tabDownload slide Bhowmick and Mugesh compared the GPx-like activity of a series of 4-methoxy-substituted N,N-dialkylbenzylamine-based diselenides with their corresponding 6-methoxy-substituted compounds.231 The comparison indicated that the activity depended substantially on the position of the methoxy substituent, because all 6-methoxy-substituted diselenides 87–89 (Chart 22) exhibited much better activity than the corresponding compounds with the methoxy group at the 4- (or para) position. While, both 4- and 6-methoxy substitutions are expected to have similar electronic effects (i.e., increase the electronic density of the aromatic ring) in the selenium center of the ring, the substitution at the 6-position provided optimized steric hindrance for some key intermediates, which enhanced the catalytic activity of 6- over 4-substituted diselenides. At face value, the authors had already shown that the replacement of tertiary or tert-amino groups in benzylamine-based diselenides by secondary or sec-amino moieties considerably enhanced the catalytic activity of derivatives 90–93 (Chart 23).232 The sec-amino were more potent than the tert-amino moieties by more efficiently generating the catalytically active selenols. Chart 23 Open in new tabDownload slide Recently, a study with amine based diselenides demonstrated that the compound 94 (Chart 24) was ≅2-fold more active than diphenyl diselenide 53 as a GPx mimic towards hydrogen peroxide or tert-butyl hydroperoxide as substrates and thiophenol as a thiol co-substrate.233 The influence of the electronic effects on selenium by the interaction with the amine group was indicated as the main factor responsible for the high GPx-like activity of the amine based diselenides compared to diphenyl diselenide 53. The same authors examined the GPx-like activity of the imine based diselenide 95 (Chart 24) along with diphenyl diselenide 53 using hydrogen peroxide or tert-butyl hydroperoxide as the substrate and thiophenol as the thiol co-substrate.234 Compound 95 was a more efficient catalyst than diphenyl diselenide 53 with both peroxides as the substrate of the thiol peroxidase-like reaction. The authors also demonstrated that the thiol oxidase activity (i.e., the oxidation of thiols determined in the absence of peroxides) of diphenyl diselenide 53 was higher than that of compound 95 when dithiothreitol or cysteine were used as the source of thiols. It seems that in this case, the interaction of N with the intermediates of the thiol peroxidase-like cycle facilitated the decomposition of the peroxides, but hampered the futile oxidation of the thiols in the absence of peroxides. Chart 24 Open in new tabDownload slide With respect to amide-based diselenides, Bhabak and Mugesh compared the GPx-like activity of various compounds with secondary and tertiary amide substituents, using hydrogen peroxide, cumene hydroperoxide and tert-butyl hydroperoxide as the substrates and thiophenol as the thiol co-substrate.235 The diaryl diselenides 96–99 (Chart 25) with tert-amide substituents were much better catalysts than the corresponding sec-amide-based diselenides in the reduction of peroxides by thiols, decreasing the Se⋯O non-bonded interactions. However, the catalytic activities of the tert-amide-based diselenides depended on the nature of the peroxide. In comparison, the sec-amide derivatives exhibited almost identical activities in all three peroxide systems. Chart 25 Open in new tabDownload slide Recently, Bhowmick and collaborators described the synthesis and antioxidant properties of novel diselenide and isoselenazole derivatives from amide-based diaryl diselenides, including the GPx-like activity using GSH as a reducing thiol and three different peroxides as the substrates.236 Although the diselenides 100–103 (Chart 26) exhibited lower activities than those of isoselenazoles, they were marginally more active than ebselen 1. In particular, compounds 101–103, which had larger substituents on the nitrogen atoms, exhibited lower activities than 100. It is probable that the bulkier substituents in 101, 102 and 103 hindered the interactions with GSH, slowing down important reactions such as the cleavage of the Se–Se bond by GSH. Chart 26 Open in new tabDownload slide Findings from a study with diferrocenyl diselenides as GPx mimics showed that 81, 82 (Chart 20) and 104 (Chart 27), with the selenium atom directly bonded to redox-active ferrocenyl units, displayed stronger GPx-like activity than that of diphenyl diselenide 53.237 Among them, compound 104 was much less reactive than 81 and 82, which also contained a nearby nitrogen moiety. These results show the synergic effects of tertiary amino and ferrocenyl groups on the peroxidase activity of the compounds. Chart 27 Open in new tabDownload slide Eleven imidazole diselenides derived from ovothiols, 105–115, have been synthesized and assayed for their ability to reduce hydrogen peroxide in the presence of GSH (Chart 28).238 Compounds 110 and 112 were ≅4 times more potent than the reference compound, ebselen 1. The other diselenides showed roughly the same catalytic activities, which were two to three times more effective than ebselen. The most effective compounds were those substituted at position 5 of the imidazole ring, close to the selenium atom. The main advantage of imidazole diselenides over diphenyl diselenide 53 is related to the property of being easily reduced by GSH. Chart 28 Open in new tabDownload slide In an elegant study, Shao-Wu and collaborators developed a novel dicyclodextrinyl diselenide to imitate GPx, incorporating a cyclohexylamine group in the proximity of the Se atom to increase the stability of the nucleophilic intermediate selenolate, and a b-cyclodextrin to provide a hydrophobic environment for the binding of substrates such as hydrogen peroxide and GSH.239 As expected, the compound 116 (Chart 29) exhibited better efficacy than ebselen 1 in reducing hydrogen peroxide, tert-butyl hydroperoxide and cumenyl hydroperoxide by GSH. The superior activity was attributed primarily to two factors: (1) the substrate-binding site matches the size and shape of the substrates; and (2) the incorporation of an imido-group increases the stability of the selenolate transition state in the catalytic cycle. Chart 29 Open in new tabDownload slide Based on the interest in amino acid derivatives containing chalcogens, Alberto and collaborators evaluated the GPx-like activity of new chiral-diselenide amino acid derivatives, which were synthesized by a simple and efficient two-step route.240 Among the diselenides studied, compounds 117 and 118 (Chart 30), derived from l-phenylalanine and with longer carbon chain length between the Se atoms and the amide bonds, showed higher activity than diphenyl diselenide 53 (T50 = 45.1 min, 51.4 min and 51.8 min, respectively). In contrast, the compound with a shorter chain length was the least effective catalyst in this screen (T50 = 93.0 min). Chart 30 Open in new tabDownload slide The naphthalene peridiselenide 119 and its novel dimethoxy derivative 120 (Chart 31) were tested along with diphenyl diselenide 53 in the benzyl thiol-mediated reduction of hydrogen peroxide for comparison as GPx mimetics.241 A significant increase in the GPx-like catalytic activity of simple diaryl diselenides was achieved by exploiting the conformationally restricted peridiselenide moiety (119) and by introducing electron-donating methoxy substituents to facilitate the rate-determining Se(ii) to Se(iv) oxidation step in the catalytic cycle (120). The catalytic thiol peroxidase-like cycle of the peridiselenide 119 is shown in Fig. 9. Chart 31 Open in new tabDownload slide Fig. 9 Open in new tabDownload slide The GPx-like catalytic cycle of peridiselenides. In contrast to other diselendies, the peridiselenides do not enter the thiol peroxidase-like cycle via the formation of a selenol/selenolate intermediate. The proximity of the two Se atoms in the rigid aromatic structure does now allow the stable formation of the selenolate intermediates. The catalytic cycle involves the oxidation of one of the selenium atoms in the –Se–Se– moiety. The selenoxide intermediate formed is reduced back to the active peridiselenide by thiols. A study carried out by Soares et al.242 described the preparation and GPx-like activity of a new series of selenium compound derivatives from (−)-ephedrine, using thiophenol as a thiol cofactor. Ephedrine derivatives 121–123 (Chart 32) showed powerful GPx-like activity, reducing hydrogen peroxide to water faster than diphenyl diselenide 53. The efficacy of ephedrine derivative 123 was associated with the labile selenoester functional group and the formation of the corresponding selenolate in situ, which substantially impacted its activity. The efficiency of compound 121 was attributed to interactions between selenium and the nitrogen moiety in the course of the reaction. Chart 32 Open in new tabDownload slide The GPx-like activities of water soluble cationic diselenide derivatives of benzimidazolin-2-selenones and imidazolin-2-selenones have been examined by Manjare et al.243 The GPx-like activities of cationic diselenides were found to be quite low when compared to the standard diphenyl diselenide 53. The low GPx-like activity exhibited by cationic diselenides appears to be due to the formation of insoluble oxidized selenium derivatives. However, the water-soluble diselenides synthesized from selenocystine 124, namely selenocystamine 125, diselenodipropionic acid 126 and the methyl ester of diselenodipropionic acid 127 (Chart 33), exhibited high efficiency as GPx mimics.244 The GPx-like activity of these compounds was found to be in the order 124 ≅ 125 > 127 > 126. From one-electron redox studies using the nanosecond pulse radiolysis technique, the authors indicated that the high thiol peroxidase-like activity of 124 and 125 was related with their ability to undergo simple one-electron reduction. Chart 33 Open in new tabDownload slide Prabhu and collaborators synthesized and evaluated the catalytic efficiency of a new series of nicotinoyl based organoselenium compounds as GPx mimics, using methods based on 1H-NMR spectroscopy and HPLC.245 Compounds 128 and 129 (Chart 34) showed the highest GPx activity, exhibiting better activity than ebselen 1. The authors confirmed that the GPx-like activity of nicotinamide based organoselenium compounds depended upon the nature of the substituent attached to the nitrogen atom of the amide group, because the introduction of either electron donating groups, such as phenyl, or electron withdrawing groups, such as pyrimidine, to the amide linkage significantly reduced the GPx-like activity. From 77Se NMR studies on the intermediate species formed in the GPx-like cycle, the authors identified that after the reduction by GSH, compound 129 was converted to a stable selone, which was likely responsible for its efficient GPx-like activity.246 The catalytic cycle of the nicotinoyl based organoselenium compound 128 is depicted in Fig. 10. Chart 34 Open in new tabDownload slide Fig. 10 Open in new tabDownload slide The thiol peroxidase-like cycle of nicotinoyl based organoselenium compounds. The compound 128 is shown as a prototypal compound of this sub-class of diselenide compound. The catalytic cycle of 128 involves the formation of selenol/selenolate intermediates, but due to electronic effects, the selenol is transformed in the selone intermediate (–C˭Se), which reduces the peroxides in the catalytic cycle. Analogous to other simple diselenides and to native GPx, the oxidation of the selenium atom of the selone forms the selenenic acid intermediate. Given the role of pyridoxine as an antioxidant in biological systems, the Singh research group developed and examined the thiol peroxidase-like activity of a number of new pyridoxine-derived organoselenium compounds.247 Among the compounds assessed in an aqueous solution, diselenide 130 (Chart 35) showed higher GPx-like activity than ebselen 1. Subsequently, the authors tested the effect of a pyridoxine-derivative diselenide carrying an extra bromine in position 6, 131 (Chart 35).248 Diselenide 131 showed a reactivity 3-fold higher than ebselen 1. As the bromine turns the pyridylseleno group into a better leaving group, the nucleophilic attack of thiol (GSH) on the selenium atoms in the diselenide 131 was greatly facilitated and might be the key to understanding the high GPx-like activity of this compound. Chart 35 Open in new tabDownload slide Cholesterol-containing diselenides have been investigated as mimetics of GPx. For instance, dicholesteroyl 132 (Chart 36) exhibited negligible thiol peroxidase-like activity, probably because the bulky moiety of the dicholesteryl 132 sterically hindered the –Se–Se– bond from interacting with thiols.249 Accordingly, the dicholesteryl 132 did not oxidize dithiothreitol and other thiol-containing molecules in the absence of peroxides (thiol oxidase assay). Chart 36 Open in new tabDownload slide Recently Frizon and coworkers reported the synthesis and GPx-like activity of a new series of chalcogen liquid crystal containing dicholesteroyl diselenide moieties in their structures.250 All prepared diselenides showed good thiol peroxidase-like activity using benzenethiol as a GSH alternative. However, the derivative 133 had higher activity than ebselen 1 (about 3.4 times). The differences in the chemical behavior of the dicholesteroyl derivatives 132 and 133 may be explained by the fact that in 132 the Se atoms are bound directly to the bulky cholesteroyl moieties, whereas in 133 the Se atoms are bound to much less hindered aliphatic segments. Rafique and collaborators studied the potential antioxidant properties of a new series of 2-picolylamide-based diselenide with O⋯Se non-bonded interactions and all the compounds were effective GPx mimics.251 However, the aromatic 2-picolylamide diselenide derivative 134 (Chart 37) presented stronger thiol peroxidase-like activity when compared to other aliphatic 2-picolylamide diselenide derivatives and the standard diphenyl diselenide 53. The authors suggested that the Se⋯O interaction in compound 134 may promote the easy conversion of selenyl sulfide to selenol/selenolate, a fundamental step in the GPx-like catalytic cycle. Chart 37 Open in new tabDownload slide The GPx-like activity of new aliphatic and aromatic nitrogen-containing diselenides synthesized by Nascimento and coworkers was evaluated by their ability to reduce hydrogen peroxide to water at the expense of thiophenol.128 The best results were obtained with the aromatic derivatives 135 (T50 = 16.8 min) and 136 (T50 = 29.7 min) (Chart 37), which were more active than ebselen 1 (T50 = 154.2 min) and diphenyl diselenide 53 (T50 = 55.0 min). The improved thiol peroxidase-like activity of the derivatives was attributed to the non-covalent interaction between Se and the amidic nitrogen. The structural constraint forced such interactions to occur and enhanced the GPx-like activity, particularly in the aromatic derivatives. Novel symmetrical diselenides and their selenocyanate analogues have been synthesized and their GPx-like activity determined.252 Among them, compound 137 (Chart 38) exhibited the most potent thiol peroxidase-like activity, which was ≅2 times greater than that of ebselen 1. In contrast, ebselen derivatives (ebselen selenocyanate and ebselen diselenide) were not active as GPx mimetics. This differential behavior among compounds was attributed to the electronic nature of the substituents at the phenyl groups in the para position that, contrary to expectation, diminished the GPx-like activity. Chart 38 Open in new tabDownload slide Selenium compounds as mimetics of iodothyronine deiodinase As stated in the introduction, the human genome codifies 25 selenoproteins, including the 3 isoforms of iodothyronine deiodinase (D1, ID-1 or DIO1, D2, ID-2 or DIO2 and D3, ID-3 or DIO3). Deiodinases catalyze the reductive dehalogenation of iodothyronines; however, they have different specificity and affinity for the substrates (i.e., 3,5,3′,5′-tetraiodothyronine or T4, 3,5,3′-triiodothyronine or T3, and 3,3′,5′-triiodothyronine, reverse T3 or rT3), tissue and cellular localization.253–258 These enzymes are important in the metabolism of thyroxine (T4), which is the main thyroid pro-hormone in vertebrate. T4 is activated by 5′-deiodination in the outer ring of the T4 molecule and can be catalyzed by either DIO1 or DIO2. The product of this reaction is the metabolically active hormone T3 (Fig. 11). DIO2 is expressed in several tissues and is located in the endoplasmic reticulum. DIO3 is a plasma membrane protein involved in the deactivation of T3 and can cause the deiodination of T4 (at the tyrosyl ring) to form an inactive isomer of T3 (rT3). DIO3 can inactivate T3 by catalyzing its inner-ring deiodination, which forms the inactive metabolite 3,3′-diiodothyronine or T2 or 3,3′-T2. The details about the structure of DIO isoforms are still elusive. More recently, the three dimensional structure of the active center of DIO3 was characterized in detail.259 Thus, although the three isoforms of DIO share some basic structural features, particularly in their primary structures near the active site,257,258,260,261 little is known about the subtle differences in the structures of the isoforms. The understanding of such differences will be important to elucidate the catalytic mechanism(s) of DIO isoforms in detail, including the molecular basis of substrate specificity and affinity. The elucidation of the DIO3 crystal structure showed that the selenocysteine (U170) is in a hydrophobic bulky position close to the prolinyl residues 171 and 172 and the Phe258 (Fig. 12).259 Fig. 11 Open in new tabDownload slide Reaction mechanism of iodothyronine deiodinases (A) and the respective reactions of each isoform (B). Fig. 12 Open in new tabDownload slide The active site of DIO3 (PDB 4TR4). The U170 interacts with hydrophobic residues (P171, P172 and F258). The H-bond network involved in the catalysis is indicated with green lines. Of particular pharmacological and therapeutic importance, detailed knowledge about the catalytic properties of DIO isoforms will be important to guide the development of effective compounds to treat either hypothyroidism or hyperthyroidism. Here we have performed molecular docking between DIO3 and the active and inactive thyroid hormone metabolites in order to detect subtle differences in their interaction with DIO3. As we can see in Fig. 13, the ID-3 substrates T4 and T3 (Fig. 13A and B) presented the iodine atom of the tyrosyl ring close to the selenocysteinyl (U170) (3.9 Å). The proximity of the selenol group of U170 to the iodine atom may facilitate the nucleophilic attack by the selenolate group on the halogen, causing the deiodination of the substrates. On the other hand, the ligands rT3 and 3,3′-T2 (Fig. 13C and D) (that are not substrates of ID-3) apparently did not display Se⋯I interactions, because of the conformation of the tyrosyl ring. It is important to observe that the outer ring of the thyroid hormones (T4, T3, rT3 and 3,3′-T2) are far from the U170, interacting with the prolinyl residue (P171) by σ–π stacking and H-bonding with hytidinyl 202 (H202). In addition, the amino and carboxylic acid groups of the ligands made H-bonds with G276, R275 and E259, and these residues probably guide the correct conformation in the active site of the enzyme, besides the phenylalaninyl residue (F258), that makes the π–π stacking with the tyrosinyl ring of the substrates. Fig. 13 Open in new tabDownload slide Interactions between ID-3 and T4 (A), T3 (B), rT3 (C) and 3,3′-T2 (D). Molecular docking simulations were performed with the AutoDock Vina 1.1.1 program, using a gridbox of 20 × 20 × 30 Å (coordinates: −10.159; −1.433; 16.492) centered on the active site of ID-3 (PDB: 4TR4).259 The Cys170 was replaced to Sec170, with the Se atom deprotonated. The Se partial charge (−0.821) was calculated by semi-empirical PM6 single point calculation, using the dielectric constant of water with the MOPAC program, in a system containing residues within 4 Å of the Se atom. During the docking, the lateral chain of the Phe258 residue was considered flexible. The ligands were built with the Avogadro program and the geometries and charges were obtained by semi-empirical PM6 optimization with the MOPAC software. The enzymes were prepared for docking with the Chimera 1.8 program, where the water molecules were deleted and hydrogens atoms added. The results were analyzed using Accelrys Discovery Studio 3.5.85 In fact, the search for new compounds that can be selective towards specific DIO isoforms has gained interest in the literature.260,262,263 The development of inhibitors of DIO1 can be useful in the treatment of hyperthyroidism262,264 whereas the synthesis of new mimetic compounds of DIO2 will be of potential therapeutic exploitation in the treatment of hypothyroidism. Accordingly, the literature data have indicated that DIO2 and DIO3 can regulate the levels of the thyroid hormone in the cellular and subcellular environment, modulating the T3 signalling pathways in specific cells, independent of fluctuations in T3 blood levels.257,258,265 The search for mimetic compounds of DIO started about two to three decades ago.260,266 The synthesis of DIO mimetic compounds has been motivated by the interest of understanding the catalytic cycle of the DIO isoforms and to develop low molecular mass compounds that could imitate the native isoforms of DIO. For instance, the reduced form of diphenyl diselenide PhSeH (benzene selenol) has been shown to promote the deionization of 4-nitro-2,6-di-iodophenol.267 In the last two decades, Mugesh and collaborators have been exploring the potential of organochalcogens as mimics or inhibitors of DIO isoforms.263,264,268–273 Particularly, they have studied in detail naphthyl-based dithiol, thiol-selenol or diselenol compounds270–273 (Chart 39). Chart 39 Open in new tabDownload slide The superior activity of diselenol derivatives as agents capable of promoting dehalogenation of thyroid hormones and analogs was explained on the basis of co-operative action of halogen bonding (between one selenium and iodine atom) and selenium bonding (between the two peri-selenium atoms) in peri-napthalenes or peri-substituted naphthalenes.270–272 More recently, the synthesis of a stable arylselenenol group that can be transformed in the iodide intermediate, having a cavity-shaped steric protection group (150, Chart 39), was described as being capable of promoting the deiodination of T4 derivatives.274 The same approach was used by Goto and collaborators to synthesize primary-alkyl-substituted selenyl iodide (151).275 The tridimensional structure of 150 and 151 demonstrated the presence of a hydrophobic bulky cavity, which is supposed to stabilize the selenol group (Fig. 14). The cavity protected –SeH group can partially imitate the characteristic of the DIO3 active center. Fig. 14 Open in new tabDownload slide A 3D representation of compounds 150 (A) and 151 (B), in stick (left) and surface (right) models. In the surface models, the selenium cavity is highlighted. The surface color is determined by atom partial charge, where blue and red represent positive and negative regions, respectively. The structures were built with the Avogadro software followed by PM6 geometry optimization with MOPAC. Taken together, the studies reviewed here indicate the potential use of organoselenium compounds as mimics of DIO isoforms. However, as commented above for other GPx mimics, the selectivity of organoselenium compounds as a mimic of specific DIO isoforms has not been studied in detail. For instance, some of the presented compounds, e.g., the peri-substituted naphthalenes having different amino groups, promote the specific removal of the iodine atom from the inner-ring of T4 and T3 to produce rT3 and 3,3′-T2. These reactions are similar to those catalyzed by DIO3 and DIO1 (the reaction forming rT3), and consequently the peri-naphthalenes could be exploited to reduce the levels of T3 and T4 in the blood and other tissues. In contrast, the synthesis of organoselenium compounds that could selectively catalyse the outer-ring removal of the iodine atom at the 5-position has not yet been described. The synthesis of such type of compound is highly attractive, because they could potentially be useful as therapeutic agents to treat hypothyroidism. Organoselenium compounds as mimics of other selenoenzymes As discussed above, organoselenium compounds can imitate GPx and iodothyrionine deiodinase isoforms. In contrast, we did not find examples of organoselenium compounds that are mimics of selenophosphate synthetase nor other selenoproteins. This is somewhat intriguing because any compound that can be metabolized to a selenol intermediate can hypothetically be a mimetic of some selenoproteins. Perhaps, the limitation here is how to quantify the mimetic activity of a given compound using analytically relevant assays for assessing the activity of specific selenoproteins. For the case of GPx isoforms, the available analytical methodologies are simple and involve spectrophotometric methodologies (see below). The simplicity of following the oxidation of different thiol substrates has allowed the comparison of the activities of the synthetic compounds with the native enzyme.228,276 For the case of deiodinase isoforms, the methodologies are more complex and there is no study comparing the catalytic efficiency of the compounds with the native enzymes. Consequently, the standardization of analytical methodologies that can compare the relative efficiency of synthetic compounds with those of native isoforms of DIO are important to determine which type of selenium compound could be effective as a mimetic of deiodinase enzymes. For the case of selenophosphate synthetase, the basic reaction should be carried out between the selenium compounds and ATP, and the formation of selenophosphate quantified. However, no data in the literature has given indications that this type of reaction will occur. In the case of methionine sulfoxide reductase (MsrB1), the reaction of selenol intermediates with methionine-sulfoxide or other model sulfoxide molecules is plausible. In fact, one of the putative mechanisms of sulfoxide reduction involves nucleophilic attack by the –SeH group (in the selenolate form) on the oxidized sulfur atom of methione sulfoxide to form a tetrahedral intermediate, which subsequently decomposes to form a selenenic acid intermediate and reduced methionine. The selenenic acid, in a similar way to that which occurs in the GPx-like cycle, can be reduced back to the selenol intermediate by reacting sequentially with 2 equivalents of thiol groups.277 In the case of more complex selenoenzymes, for instance thioredoxin reductase, the synthesis of mimetic organoselenium compounds will be more difficult, because they will have to possess selenol and thiol groups in complex spatial disposition similar to those found in the active asymmetric dimer of active TrxR.278 TrxR has, in the carboxy-terminal portion, the active selenol center (-Gly-Cys-Sec-Gly-OH) and a second set of redox active dithiol/disulfide parts in the N-terminal region of the enzyme (i.e., the sequence -Cys-Val-Asn-Val-Gly-Cys-). Ideally, a good TrxR mimetic will have to have these two redox centers in the same molecule. The thiol modifier effects of organoselenium compounds: alternative pathways relevant to their antioxidant properties Evidence reported over the years has revealed a myriad of biological and pharmacological properties of organochalcogen compounds including antioxidant, anti-inflammatory, neuroprotective, antiatherosclerotic and anti-cancer effects.224,279 These therapeutic properties of organoselenium compounds have been attributed to their ability to mimic the enzymatic properties of GPx.30 The first and most prevalent GPx mimetic is undoubtedly ebselen, whose peroxidase activity has been evaluated with various peroxide substrates including peroxinitrite.22,51,280,281 As discussed in the previous sections of this review, various synthetic molecules containing selenium have been designed to mimic the peroxidase activity of GPx. As an example, the diselenides designed by Spector's group are easier to synthesize and display a higher apparent thiol peroxidase-like activity when compared to ebselen.228 Despite displaying a catalytic mechanism analogous to that of GPx, the kinetic rates of these synthetic compounds are 3 to 5 orders of magnitude lower than the values calculated for the GPx enzyme, indicating the superiority of native GPx in detoxifying peroxides over synthetic small molecules.276 Orian and Toppo137 recently published a very interesting critical review pointing out the features of this class of compound as GPx mimetics. The authors discussed some of the peculiarities of organoselenium compounds, and suggested that despite their interesting mechanistic features, these drugs cannot act efficiently as enzymes given that: (i) they are not substrate specific; (ii) their selenium atom is not protected as in a catalytic center of macromolecules; and (iii) their degradation products in vivo may be reactive. Mounting evidence has indicated that the biological and pharmaceutical properties of organoselenium compounds are much more complex than expected and go far beyond their GPx mimetic activity61,224 In this sense, other mechanisms must play a role in the remarkable effects of organochalcogens in several models of diseases. In a biological system, as in the cellular environment, the organoselenium compounds to react with thiols, and, consequently, consume thiols to be activated. Thus, they behave as weak electrophiles,282 and, only after their reduction will they react as nucleophiles and can exert the peroxidase mimetic activity. The balance between these two reactions drives their biological role as oxidants or antioxidants.137 Being good weak electrophiles, these organochalcogen compounds can oxidize not only cysteine, GSH and thioredoxin, but also other redox-sensitive thiols.10,124,125,282 With a few exceptions, the weak electrophilic characteristics of organochalcogens are attributed to their selenium atom being covalently bonded as R–Se–R or as R–Se–Se–R, which may be opened to the selenol intermediate after reacting with thiols. Positive evidence for the effect as a “thiol modifier”, culminating in cellular adaptive stress responses, was firstly reported for ebselen and subsequently extended to other organochalcogens, such as diphenyl diselenide and some diselenide derivatives.276,282–285 The activation of the Keap1/Nrf2 signaling pathway via the oxidation of critical cysteinyl residues in Keap1 allows Nrf2 to transcriptionally activate the expression of antioxidant enzymes and detoxification systems.137 In support of the hypothesis that the therapeutic effects of organoselenium compounds are driven by their “thiol modifier” effect, we showed a significant improvement in the cellular antioxidant capacity via diphenyl diselenide-induced Nrf2 activation.279,284 Ebselen also induced the activation of Nrf2, but this was temporally delayed when compared with diphenyl diselenide.284 In addition, ebselen did not modify the levels of GSH and gamma-glutamyl synthetase. In this case, diphenyl diselenide promoted an efficient upregulation of the GSH system by increasing the expression and activity of GPx and gamma-glutamyl synthetase and the GSH levels, which, in turn, protected endothelial cells from nitroxidative stress induced by peroxynitrite. In contrast to an earlier study with ebselen,286 the protective effect of diphenyl diselenide against peroxynitrite cytotoxicity could not be explained by a “scavenger”-like effect or a “direct reaction” between the selenol intermediate of diselenide with peroxynitrite, since the second-order rate constant of peroxynitrite and selenophenol (the selenol intermediate formed by reduction of diphenyl diselenide) was also much lower than that reported for ebselen.284 The hypothetical interaction of diphenyl diselenide and ebselen with Keap1 is shown in Fig. 15. Fig. 15 Open in new tabDownload slide Interactions of the Keap1 protein with ebselen and diphenyl diselenide in silico.290–300 (A) Interactions of ebselen with C288 in the Keap1 protein and (B) the nucleophilic attack of C288 at the electrophilic Se atom in ebselen, forming a Se–S bond. (C) Interactions of diphenyl diselenide with C288 in the Keap1 protein and (D) the nucleophilic attack of C288 at one of the Se atoms in diphenyl diselenide, forming a Se–S bond. (E) A disulfide bond can be formed after the nucleophilic attack of C273 at the electrophilic sulfur atom of C288 (after the binding of ebselen or diselenide, i.e., B and D). (F) The reduced C273 and C288 (i.e., before interacting with organoselenium compounds or other electrophiles). A change in the secondary structure occurs when the cysteinyl residues C273 and C288 are oxidized forming a disulfide bond (note that in E and F only the fragment between the amino acid residues 240 to 314 is shown). The 3D structure of Keap1 was built with the SWISS-MODEL server,290–292 using the sequence of amino acids of the Keap1 protein from Mus musculus obtained from PubMed (https://www.ncbi.nlm.nih.gov/pubmed) (GenBank = BAA34639.1) and the 3D template from the PDB: 4AP2.293,294 First, blind docking (AutoDock Vina 1.1.1)295 with the whole 3D structure of Keap1 was performed. The region close to C288 was the preferred site for the binding of organochalcogen ligands. Next, a second molecular docking was performed restricting the interactions of chalcogens to a gridbox of 15 × 15 × 15 Å, centered on the sulfur atom of C288. The Se–S bond was built with the Avogadro program using the coordinates from the docking results.296 Residues located up to 4 Å from the Se and S atoms were considered in the analysis. The Se–S bonds were created manually and the system was optimized using the UFF force field with 2000 steps of optimization. The backbone of the Keap1 residues was frozen during the optimization processes. For the disulfide bond formation between C288 and C273, the residues from 270 to 291 were optimized with the Avogadro program, using the MMFF94 force field.297–300 In this case, 5000 steps of optimization were run, with the backbone of residues 270 and 291 frozen. Before the optimizations, the respective hydrogen atoms were added. For a better visualization, only the main residues involved in the interactions (green dotted lines) are shown. The non-polar hydrogen atoms are hidden. Bearing in mind that the reaction of GPx with peroxynitrite has been considered a biologically efficient pathway of peroxynitrite detoxification in vivo,287 the diphenyl diselenide-induced upregulation of the intracellular GSH system via Nrf2, may represent a mechanism of protection against peroxynitrite. Moreover, diphenyl diselenide is also able to increase the expression of manganese superoxide dismutase (MnSOD) and different isoforms of peroxiredoxins (Prx1 and Prx3), which represent the principal peroxynitrite detoxification pathway in vivo.285 Previously, ebselen was also reported to activate the expression of antioxidant response elements (ARE), which regulate the expression of antioxidant genes of the Prx family.288 Most interestingly, these points corroborate that diphenyl diselenide can also activate other intracellular signaling pathways. For instance, the family of transcription factor FoxO, which stimulates the transcription of gene coding for antioxidant proteins located in different subcellular compartments, such as in mitochondria (i.e. MnSOD and Prx3).289 Substantial evidence attributed the myriad of protective effects of organochalcogens to their anti-inflammatory actions. We and others showed several anti-inflammatory modulations induced by organochalcogens.301–303 Treatment with low doses of diphenyl diselenide decreased vessel-wall infiltration of inflammatory cells and the production of chemoattractant molecules, which in turn prevented atherosclerosis in a model of hypercholesterolemia in mice.301 In isolated macrophages, this organochalcogen prevented several pro-inflammatory pathways triggered by oxLDL, such as iNOS expression, metalloproteinase induction and nitric oxide production. This anti-inflammatory effect was attributed to its ability to prevent nuclear factor-kB (NFkB) activation through its antioxidant action.302 A wide range of pro-inflammatory and pro-apoptotic pathways are regulated by NFkB, which is a redox sensitive transcriptional factor. The NFkB pathways can be activated by oxidative stress because the interaction between the inhibitory protein IkB and NFkB is regulated by protein kinases that contain several redox-sensitive cysteine residues in critical kinase domains.304 We suggest that the improvement in the cellular antioxidant status and the consequent downregulation in reactive species generation promoted by diphenyl diselenide establishes a favorable cellular environment that prevents NFkB activation. Another interesting aspect regarding the promising therapeutic properties of organochalcogens lies in their ability to modulate mitochondrial function. Recent data from our research group show that diphenyl diselenide improved respiratory rates and prevented mitochondrial dysfunction in endothelial cells injured by vascular oxidants.285,305 Mitochondrial dysfunction and vascular oxidative stress alter many functions of endothelium, including the impairment on nitric oxide bioavailability via the production of the powerful oxidant peroxynitrite.306 In this way, diphenyl diselenide was able to reestablish the normal cellular bioenergetics of endothelial cells exposed to peroxynitrite or LDLox, preserving endothelial integrity.285,301 This modulation of mitochondrial metabolism is commonly correlated with an increase in the number of mitochondria. The activation of transcriptional factors such as Nrf2 and FoxO were identified as regulatory mechanisms involved in the expression of numerous genes required for the expression and function of mitochondrial respiratory chain enzymes.307,308 Alternatively, diphenyl diselenide can improve the intracellular antioxidant status by promoting GSH synthesis and the upregulation of important antioxidant enzymes such as GPx, Prx and MnSOD. The positive modulation of antioxidant elements is expected to cause a decrease in reactive species production, thereby protecting proteins and other macromolecules from oxidative damage. Therefore, the biological effects of organochalcogens are not substrate or target specific. Reactive molecules derived from the metabolism of organochalcogens, as well as the selenol intermediates, can interact with different types of redox sensitive disulfide-thiol center present in the cell, which can cause a wide range of beneficial or pathological effects associated with these compounds.10,136,137,309,310 A schematic indication of how organoselenium compounds are transformed via a reactive selenol intermediate, which reacts with oxygen to form either superoxide, a selenyl radical or diselenide, is presented in Fig. 16. The net result of formation of such reactive selenol can be the formation of reactive species and the “futile oxidation of thiol groups”. Fig. 16 Open in new tabDownload slide Oxidation of thiol and selenol groups under aerobic conditions. Although the reaction of thiols with organoselenium compounds is critical to the formation of reducing selenol intermediates, the reaction of selenol with O2 can result in the formation of toxic intermediates, such as the superoxide and selenyl radical. Furthermore, the catalytic oxidation of thiols can also have toxic consequences to the cell. Sparing the selenoproteins from the toxic electrophilic mercury forms The above sections have shown that various diorganyl diselenides and some diorganyl selenides can be transformed into selenol intermediates after interacting with reducing thiols. For instance, diphenyl diselenide and ebselen can be reduced by thiols to selenol/selenolate intermediates and the selenol/selenolate intermediates may play an important role in the antioxidant properties of both molecules. Although the formation of selenol intermediates of organoselenium compounds can be easily demonstrated in pure chemical systems, the detection of selenol intermediates in vivo has proven to be difficult due to the high reactivity of the selenol group. Thus, the in vivo demonstration of selenol intermediates derived from organoselenium compounds remains elusive; however, the presence of selenol intermediates of diphenyl diselenide, ebselen and other related molecules can be inferred from their pharmacological effects.136 The limitations of the available analytical methods in detecting the formation of selenol intermediates in vivo or in ex vivo samples is perhaps the most critical bottleneck in the study of the pharmacological, toxicological and therapeutic properties of organoselenium compounds. Despite the technical limitations for demonstrating the formation of the selenol group from organoselenium compounds inside living cells, we have obtained indirect evidence that diselenide can be transformed to the selenol/selenolate intermediate and can react with methylmercury (CH3Hg+) in a stable way. Specifically, mice were treated perorally (p.o.) with CH3Hg+ and subcutaneously (s.c.) with diphenyl diselenide for 5 weeks.311 The administration of CH3Hg+ increased the deposition of total Hg in the cerebrum, cerebellum, liver and kidney, whereas the simultaneous exposure to CH3Hg+ and diphenyl diselenide caused a marked decrease in the deposition of Hg in all tissues studied. In another study with mice, CH3Hg+ poisoning via drinking water caused an increase in brain Hg deposition (detected by autometallographic histological methods) and diphenyl diselenide (s.c.) markedly reduced the Hg deposition.312 Several biochemical endpoints of toxicity were altered by CH3Hg+ and diphenyl diselenide blunted part of the biochemical alterations caused by CH3Hg+ in both studies.311–313 We explained the decrease in the brain, liver and kidney burden of Hg by the formation of a more excretable complex of CH3Hg+ with the selenol/selenolate intermediate of diselenide (Fig. 17). The complex CH3HgSePh is expected to be chemically inert and much less toxic than CH3Hg+. Fig. 17 Open in new tabDownload slide The interaction of the selenol/selenolate intermediate (PhSe−) from diphenyl diselenide (PhSeSePh) with methylmercury (CH3Hg+). Diphenyl diselenide (PhSeSePh) can be metabolized to its selenol/selenolate intermediate (PhSe−) by a direct interaction with thiols (GSH) or via reduction by the action of TrxR. The selenolate (PhSe−) attacks the electrophilic methylmercury, forming a stable and inert complex that can be excreted more efficiently than methylmercury. The blue color indicates electropositivity, whereas the red color indicates electronegativity of the regions of the selenium compounds and methylmercury. The structures of the compounds were built with the Avogadro296 software followed by semi-empirical PM6 geometry optimization, using the dielectric constant of water. The formation of complexes between CH3Hg+ and HSe− has been demonstrated in different species.314–322 In chemical models, the binding of selenide to CH3Hg+ accelerated the breakage of the C–Hg bond. The final product containing mercury was the inert HgSe salt.323 Since diphenyl diselenide and other selenides can be metabolized to selenide324,325 there are two pathways for interaction between diphenyl diselenide and CH3Hg+: (a) the direct interaction of CH3Hg+ with the diphenyl diselenide selenol intermediate (Fig. 17) and (b) the interaction of CH3Hg+ with the selenide released from the organic moiety of diselenide. In contrast to mice, the treatment with CH3Hg+ (p.o.) and diphenyl diselenide (i.p.) caused an increase in the deposition of Hg in the brain and liver of rats.326 The increase was possibly mediated by the release of selenium from diphenyl diselenide, which can be metabolized to selenide (HSe−) after the breaking of the C–Se bond in the mono- and diselenides.324,325 The interaction of mercury with inorganic selenide has been reported in the literature and the formation of precipitates containing complexes of mercury and selenide has been reported in different tissues.314–322 The complexes are expected to be less toxic than the mercurial and explain the early observation showing the protective effects of selenium against mercury, even after an increase in the deposition of Hg induced by selenium in organs such as the brain.315,327,328 Indeed, Hg2+ and CH3Hg+ have much higher affinities for hydrogen selenide (HSe−) and selenol/selenolate (R-SeH/R-Se−) than for thiols (for reviews see ref. 329–332). As a corollary, the affinity of the electrophilic forms of mercury to the selenoproteins should be expected to be higher than that for thiol-containing proteins.329–333 The protective effects of diphenyl diselenide and ebselen against CH3Hg+ have also been demonstrated in different in vitro models of neurotoxicity.334–343 The neurotoxicity of CH3Hg+ was antagonized by ebselen after in vivo administration in rodents;344,345 in contrast, ebselen increased the hepatotoxicity of CH3Hg+ in adult mice.346 Unfortunately, Hg levels were not determined in these studies and, consequently, the reduction in Hg body burden could not be ascertained. However, considering the inclusion of ebselen as a safe therapeutic agent in the National Institutes of Health Clinical Collection32 and given its low toxicity in humans, we suggested that ebselen should be considered as a promising agent to treat CH3Hg+ poisoning in humans. Accordingly, the interaction of ebselen with methylmercury and the reestablishment of normal selenoprotein synthesis were demonstrated in vitro.347 The mechanism of ebselen protection against CH3Hg+ involves its metabolism to its selenol intermediate that binds to methylmercury, forming a stable complex of the type described for diphenyl diselenide (Fig. 17). Of particular toxicological significance, the interaction of mercurials with selenoproteins has been described by different laboratories.347–354 The high affinity of purified TrxR for both Hg2+ and CH3Hg+ was studied in detail and the purified enzyme was inhibited by nanomolar concentrations of both mercury forms352,353 and selenide (formed from selenite reduction) protected the TrxR from both forms of mercury.353 Accordingly, the inhibition of selenoenzymes by mercury has also been observed in crude cell extracts355 or with partially purified TrxR from different tissues of mice.350 The TrxR system of rodents and fish has also been targeted by CH3Hg+ after in vivo exposure, but only few studies have investigated the protective effect of selenium.335,355–358 Some hypothetical interactions between CH3Hg+ (MeHg and complexes) and Hg2+ with TrxR1 are depicted in Fig. 18. Fig. 18 Open in new tabDownload slide Interactions between the MeHg+ and Hg2+ complexes with TrxR-1 in silico. (A) The interactions between TrxR-1 and MeHg+ complexed with one molecule of water [MeHgH2O]+ and (B) complexed with cysteine, and (C) the formation of the Se–Hg bond after the nucleophilic attack by the Se atom (in the Sec residue (U498)) at the Hg atom in the MeHg molecule. (D) The interactions of Hg2+ (as the complex of the type [H2O–Hg–OH]+ 359) with the active site of TrxR-1, indicating the formation of –Se–Hg–S– bonds, involving the Sec (U498) and Cys (C497) residues. (E) After the nucleophilic attack of the Se and S atoms at the electrophilic Hg atom, two molecules of water are released. The interactions depicted in A, B and D were obtained from the molecular docking calculations (AutoDock Vina 1.1.1),295 using a gridbox of 15 × 15 × 15 Å and centered on the sulfur atom of C498 of TrxR-1 (PDB: 2J3N). The sulfur atom of C498 was replaced by the selenium atom to form the actual U498 residue of Human TrxR-1. The atomic charge of selenide (−0.834) in U498 was obtained by semi-empirical PM6 single point calculation using the dielectric constant of water with the MOPAC program.360 Only residues with a distance of up to 4 Å from the Se atom were considered in the optimization, to find the Se charge. The Se–Hg complexes depicted in C and E were built with the Avogadro software,296 using the coordinates from the docking results. To optimize the Se–Hg complex, the residues within 4 Å of the Se and Hg atoms were considered. The Se–Hg–S bond was created manually and the system was optimized using the UFF force field with 2000 steps of optimization. The backbone of U498 was frozen during the calculations. Hydrogen atoms were added in the calculations. The ligands were generated with the Avogadro program and the final geometry and atom charges were obtained from PM6 optimization using the dielectric constant of water (78.0) with MOPAC. The TrxR-1 protein was obtained from the Protein Data Bank (2J3N)361 and prepared for the docking studies with the Chimera 1.8 software, using the chains C and D. The ligands and protein in the pdbqt format were generated by AutoDockTools, where the ligands were considered flexible, and the enzyme rigid (with Gasteiger charges).362 For the docking simulation, in the pdbqt file the Hg atom was replaced by Zn because the AutoDock Vina program did not present parameters for the Hg atom. The results were analyzed using Accelrys Discovery Studio 3.5.85 For the sake of clarity, the non-polar hydrogens are hidden and only the main residues involved in the interactions (green dotted lines) are shown. The residues in the active site present the carbon atoms in pink. CH3Hg+ was also reported as a weak inhibitor of GPx in vitro; the difference in relation to TrxR can be explained in terms of the reactivity/accessibility of the selenol groups of the two enzymes (Fig. 18 and 19). Despite the low sensitivity of GPx to CH3Hg+in vitro, the literature has indicated that the different isoforms of GPx are decreased after the exposure of cells or vertebrates to CH3Hg+.311,349Fig. 19 depicts hypothetical types of interaction of CH3Hg+ (MeHg) and MeHg complexes with GPx1 simulated in silico. Fig. 19 Open in new tabDownload slide Interactions of CH3Hg+ (MeHg) and MeHg complexes with GPx1 in silico. The interactions of MeHg alone (A, top left), the MeHg+–H2O complex (B, top right) and the MeHg+–cysteine (C, bottom left) complex with the selenocysteinyl residue of GPx1 (Sec45 or U45) were obtained from molecular docking with the AutoDock Vina 1.1.1 program,295 using a gridbox of 15 × 15 × 15 Å and centered on the Se atom of U45 of GPx-1 (PDB: 1GP1).76 To generate the selenocysteine residue in the docking studies, the oxygen atoms from the seleninic acid form of the U45 residue were deleted and the selenide atom charge considered was −0.833. The charge was calculated with the semi-empirical PM6 single point calculation, using the dielectric constant of water with the MOPAC program,360 in a system containing residues within 4 Å of the Se atom. Physiologically, the most abundant complexes of MeHg are expected to be those with cysteine or with GSH, because these are the two most abundant low mass –SH groups found in vertebrates. After the formation of a stable bond between U45 and MeHg (D), the GPx1 is inactivated. The –Se–Hg– complex formed in D was built with the Avogadro software,296 using the coordinates from the docking results and considering the residues located up to 4 Å from the Se and Hg atoms in the optimization steps. The hydrogen atoms were considered in the calculations. The Se–Hg bond was created manually and the system was optimized using the UFF force field with 2000 steps of optimization, with the backbone of U45 frozen. The ligands (MeHg+ and MeHg complexes) were generated with the Avogadro program and the final geometry and atom charges were obtained from PM6 optimization using the dielectric constant of water with MOPAC. The ligands and protein in the pdbqt format were generated by AutoDockTools, where the ligands were considered flexible, and the enzyme rigid (with Gasteiger charges).362 The results were analyzed using Accelrys Discovery Studio 3.5.85 For the sake of clarity, the non-polar hydrogens are hidden and only the main residues involved in the interactions (green dotted lines) are shown. The residues in the active site present the carbon atom in pink. In contrast, human exposure to environmental or dietary CH3Hg+ has been associated with no alteration or with an increase in the levels of GPx and selenoprotein P SelPPP. The increase in SelPP levels was interpreted as an adaptation to an increased demand for selenium for selenoprotein synthesis in different tissues.363 Consistent with the important role of selenoproteins as targets of methylmercury, recent studies have pointed out changes in the expression of GPx and other selenoproteins after exposure to MeHg.364 Considering the data presented in the previous sections of this review and the interaction of organoselenium with electrophilic forms of Hg, we posit that organoselenium compounds exert their pharmacological/therapeutic or toxicological effects by diverse mechanisms (Fig. 20). Fig. 20 Open in new tabDownload slide Biochemical mechanisms involved in the protective effects of organoselenium compounds. The reduction of organoselenium compounds by thiol-containing molecules can generate selenol intermediates (R-Se−), which react with toxic electrophiles (for instance, methylmercury or CH3Hg+) or decompose toxic peroxides (thiol-peroxidase-like activity). Furthermore, the oxidation of thiol-containing proteins can activate antioxidant response elements (for instance, Keap1/Nrf2). Knowledge about the contribution of each pathway to the pharmacological properties of organoselenium compounds remains elusive. Perspectives and concluding remarks The data presented in the previous sections of this review indicate that the pharmacological and therapeutic effects of simple organoselenium compounds cannot be explained only by their chemical ability to mimic GPx activity. The interaction with thiol-containing proteins may be even more important than their capacity to mimic selenoproteins given the strong reactivity and very transitory stability of the selenol groups of low molecular mass compounds. For decades, the potential therapeutic use of organoselenium compounds has attracted the interest of chemists and biologists; however, ebselen is the only compound that has been considered a safe agent.32,34–39 Ironically, the compound does not have a specific target disease. It was used in the past in clinical trials for the treatment of brain ischemia/stroke,23–25 but its clinical use was not approved by Japanese authorities.27 At present, ebselen is considered a potential lithium mimetic, because it can inhibit a key enzyme involved in the genesis of bipolar disorder.32–36 In fact, ebselen can inhibit the IMPase by oxidizing the cysteinyl residue 218 (C218) (Fig. 21 depicts the in silico interaction of ebselen with this critical cysteinyl residue). In addition, ebselen was used in clinical trials as a potential therapeutic agent against noise-induced hearing loss37,38 and in a small number of patients with diabetes.39 Fig. 21 Open in new tabDownload slide Interaction of ebselen with 5′-IMPase. (A) Molecular docking indicating the interactions of ebselen with the IMPase (PDB: 2BJI).367 (B) Se–S bond formation by the nucleophilic attack of C218 to the electrophilic Se atom of ebselen. For the docking simulation, the I68, S79, I88, M246, D274 and C218 residues were considered flexible, with a gridbox of 10 × 10 × 10 Å centered on the sulfur atom of C218. The other docking and optimization parameters were the same as described in the legends of Fig. 15, 18, 19. For the sake of clarity, the non-polar hydrogen atoms are hidden and only the main residues involved in the interactions (green dotted lines) are shown. The residues in the active site are represented with the carbon atoms in pink. However, treatment with ebselen for 4 weeks did not improve the vascular function in diabetes type 1 or type 2 patients.39 In the case of hearing loss, a recent study indicated a very narrow therapeutic efficacy of ebselen.38 In contrast to ebselen, diphenyl diselenide did not inhibit IMPase,32 indicating some specificity of the interaction with ebselen. As commented above, the absence of specificity of organochalcogens to various types of thiol-containing protein has been considered one of the most limiting caveats in the pharmacological exploitation of organoselenium compounds. Diphenyl diselenide and ebselen can interact with other important thiol containing proteins, such as the glycolytic enzyme LDH365 (see Fig. 22) or with the enzyme porphobilinogen synthase or aminolevulinate dehydratase (δ-ALAD)366 (Fig. 23). Thus, although the interaction of organoselenium compounds with endogenous thiols is critical for their thiol-peroxidase-like activity, the absence of selectivity to interact with different target proteins can result in toxic effects. Accordingly, it will be imperative to synthesize organoselenium compounds with greater selectivity for specific target proteins. Fig. 22 Open in new tabDownload slide The interaction of ebselen and diphenyl diselenide with lactate dehydrogenase (LDH). (A and C) The interaction of ebselen (A) and diphenyl diselenide (C) with LDH (PDB: 1I10).368 (B and D) Se–S bond formation by the nucleophilic attack by the thiol group of C162 at the selenium atoms of ebselen (B) and diphenyl diselenide (D). For the docking simulation, the C162 was considered flexible, with a gridbox of 12 × 12 × 12 Å centered on the C162 sulfur atom. The other docking and optimization parameters were the same as those described in Fig. 15, 18, 19. For the sake of clarity, the non-polar hydrogens are hidden and only the main residues involved in the interactions (green dotted lines) are shown. The residues in the active site are represented with the carbon atoms in pink. Fig. 23 Open in new tabDownload slide Molecular docking between δ-ALAD and diphenyl diselenide and ebselen. (A) The interactions of diphenyl diselenide with the active site of δ-ALAD (PDB: 5HMS),369 and (B) the Se–S bond formation after the nucleophilic attack of C132 at one of the Se atoms of diphenyl diselenide. (C) The docking results for the interaction of ebselen with the active site of δ-ALAD, and (D) the Se–S bond formed between the thiol group of C132 and the Se atom from ebselen. This resulted in the opening of the isoselenazol ring. (E) The formation of a disulfide bond between the C124 and C132 residues, which resulted from the nucleophilic attack of the thiolate group of C124 at the C132 sulfur atom. For the docking simulation, the Y205, R209, R221 and Q225 residues were considered flexible, with a gridbox of 20 × 20 × 20 Å, centered on the coordinates: x = 9.2; y = 34.3; z = 73.8. The zinc ion charge (0.302) was obtained by semi-empirical PM6 single point calculation using the dielectric constant of water with the MOPAC program, considering the influence of the residues located up to 4 Å from the Zn atom. The disulfide bond formation between C124 and C132 was created manually and optimized with the Avogadro program with the UFF force field and 2000 steps of optimization, considering the residues within 4 Å of the Zn atom and the ligands (ebselen and diphenyl diselenide), with the backbone of the protein frozen. The other docking and optimization parameters were the same as those indicated in Fig. 15, 18, 19. For the sake of clarity, the non-polar hydrogens are hidden and only the main residues involved in the interactions (green dotted lines) are shown. The importance of the GPx- or thiol-peroxidase-like activity of organoselenium compounds in their pharmacological properties needs to be reconsidered, because low mass organoselenium compounds cannot congregate in their simple structure all the steric and electronic interactions found in the active site of selenoproteins (compare Fig. 2 with Fig. 24, which show some interactions between the selenium atoms and tertiary amino groups in two of the most active mimetics of GPx described in the literature,228 i.e., compounds 85 and 86 presented in Chart 21). Although the selenolate intermediates from diselenides 85 and 86 can mimic some interactions analogous to those found in the active site of GPx, they are still too rudimentary when compared to those found in the active center of selenoenzymes. Consequently, it is difficult to understand how a selenium compound that forms simple selenol/selenolate/selone intermediates can mimic only the activity of one class of selenoprotein, i.e., GPx isoforms. If simple selenium compounds can form selenol intermediates (which will have short transitory stability), the selenol/selenolate intermediate will not interact selectively with peroxides but with any group with high affinity for soft nucleophiles (for instance, disulfide bridges, electrophilic mercury and cadmium forms, etc.). Fig. 24 Open in new tabDownload slide The structure of 2,2′-diselenobis[[(N,N-dimethylamino) methyl]benzene]bis(hydrochloride salt) (compound 85) and 2,2′-diselenobis[(pyrrolidin-l-ylmethyl)benzene]bis(hydrochloride salt) (compound 86; Chart 21) and its selenol/selenolate intermediates. The interactions depicted in I to VIII are considered important either for the reduction of the –Se–Se– bond or for the stabilization of the –SeH (selenol)/–Se−(selenolate) intermediates of compound 85 and compound 86 (Chart 21) during their thiol peroxidase-like catalytic cycle. (A) The optimized structure of 85 in the N-protonated (I) and deprotonated (II) forms. (B) The optimized structure of the selenol (–SeH) intermediate of 85 (III) and the selenolate (–Se−) N-protonated (IV) form of 85. (C) The optimized structure of 86 in the N-protonated (V) and deprotonated (VI) forms. (D) The optimized structure of 86 in the selenol (Se–H) (VII) and selenolate (N-protonated) (VIII) forms. The distances are indicated with dotted green lines. The structures of the compounds were built with the Avogadro software296 followed by semi-empirical PM6 optimization, using the dielectric constant of water (78.0) with the MOPAC program.360 The results were visualized with the Accelrys Discovery Studio 3.5 software.85 Another important aspect that needs to be considered is the standardization of procedures to determine the thiol-peroxidase-like activity of the compounds. The methodology used in various studies varies considerably both in terms of the types and the concentrations of substrate (thiol reducing agent and peroxides) used in the assays (see Table S1, ESI†). Thus, it is difficult to compare the activities among the studies. In general, we have two main methods: (1) one performed in buffered water using the coupled assay with NADPH, glutathione reductase (GR), GSH, peroxides (either H2O2 or organic peroxides, e.g., tert-butyl hydroperoxide) and the GPx mimetics. The acceleration of thiol oxidation is determined indirectly by the decrease in the absorbance of NADPH at 340 nm (Fig. 25A), which is consumed to reduce the GSSG to GSH (Fig. 25). (2) A method performed in methanol, using thiophenol (PhSH) as the reducing thiol and peroxides (H2O2 or tert-butyl hydroperoxide, etc.) where the oxidized diphenyl disulfide (PhSSPh) is measured at 305 nm (Fig. 25B). Although both methods have limitations in terms of extrapolation to in vivo situations, they can give an estimation of the capacity of a given selenium compound as a potential imitator of GPx, but it would be critical to have comparative and detailed mechanistic studies using both methods. Future studies could be beneficially directed to incorporate high-throughput in vitro assays (for instance, with cell cultures) simultaneously with GPx-like studies to determine the toxicity and pharmacology of different organoselenium compounds. Without these studies, we will not be able to construct a rational framework for more efficiently evaluating which organoselenium compounds offer the most efficacious therapies both in animal models and humans. Fig. 25 Open in new tabDownload slide The quantification of the GPx- or thiol-peroxidase-like activity of organoselenium compounds. Here the two common methodologies used to determine the GPx-like activity of organoselenium compounds are schematically presented. (A) The coupled methodology is done in aqueous buffered media, where the consumption of NADPH is proportional to GSH oxidation to GSSG and can be followed at 340 nm. (B) The methodology of Iwaoka and Tomoda226 is carried out in organic media (normally in methanol, but ethanol can also be used) and the reaction is followed by the increase in absorbance at 305 nm (PhSSPh formation). In short, simple organoselenium compounds should be considered weak “mimics of selenoproteins” and not only of GPx, because structurally simple selenol groups cannot interact in a selective way with specific targets. However, some chemical/structural characteristics may render them good GPx mimic molecules (Fig. 26). Precise knowledge about the sterical and electronic relationships between the selenol groups in the active site of selenoproteins with other amino acid residues (for instance, the triad found in the active site of GPx; Fig. 2B) can give some clues to be followed by chemists during the design of new selenocompounds. Even so, the size of new and more rational molecules will have to be much bigger than that of the simple molecules available in the literature. Thus, their potential use as pharmacological agents will be limited by factors related to absorption, distribution, metabolism and excretion of molecules with molecular mass higher than 500 Da. In view of this and considering that the interaction of organoselenium compounds with some thiol-containing proteins (for instance, Keap1 and 5′-IMPase) can induce desirable metabolic changes, perhaps the best approach will be to design new simple molecules that have selectivity to specific classes of thiol-containing group. In short, the exploitation of the thiol modifier properties of simple organoselenium compounds can be a more rational pathway to be followed than to use low mass molecular structures to mimic the activity of high mass macromolecules that have been shaped by billions of years of evolution. Fig. 26 Open in new tabDownload slide General features of good GPx mimic molecules. Conflicts of interest There are no conflicts to declare. Acknowledgements The authors would like to thank the coworkers referenced in this review for the contribution of their studies. 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Crossref Search ADS Footnotes † Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mt00083a © The Royal Society of Chemistry 2017 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © The Royal Society of Chemistry 2017
TI - Organoselenium compounds as mimics of selenoproteins and thiol modifier agents
JF - Metallomics
DO - 10.1039/c7mt00083a
DA - 2017-12-14
UR - https://www.deepdyve.com/lp/oxford-university-press/organoselenium-compounds-as-mimics-of-selenoproteins-and-thiol-s1CHPRySeg
SP - 1703
EP - 1734
VL - 9
IS - 12
DP - DeepDyve
ER -