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Concept: Disulfide bond


Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the children experience a developmental regression characterized by a loss of previously acquired skills and abilities. Typically reported are losses of verbal, nonverbal, and social abilities. Several recent studies suggest that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH) reserve capacity, resulting in a compromised oxidation/reduction (redox) and detoxification capacity. Research indicates that the availability of thiols, particularly GSH, can influence the effects of thimerosal ™ and other mercury (Hg) compounds. TM is an organomercurial compound (49.55% Hg by weight) that has been, and continues to be, used as a preservative in many childhood vaccines, particularly in developing countries. Thiol-modulating mechanisms affecting the cytotoxicity of TM have been identified. Importantly, the emergence of ASD symptoms post-6 months of age temporally follows the administration of many childhood vaccines. The purpose of the present critical review is provide mechanistic insight regarding how limited thiol availability, abnormal sulfation chemistry, and decreased GSH reserve capacity in children with an ASD could make them more susceptible to the toxic effects of TM routinely administered as part of mandated childhood immunization schedules.

Concepts: Autism, Disulfide bond, Glutathione, Asperger syndrome, Autism spectrum, Cysteine, Thiol, Thiols


In this work we investigated the formation, reactivity and anti-platelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the ten thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol (DFT), and 3-nitropyridine-2-thiol (NPT). The MS and MS2 spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M(-1)s(-1). The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pre-treatment with 1 mM GSH, confirming that the AM is responsible for the anti-platelet activity of clopidogrel. Collectively, our results provide strong support for a P450-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored in order to overcome the inter-individual variability in clopidogrel therapy.

Concepts: Platelet, Disulfide bond, Glutathione, Sulfur, Cysteine, Thiol, Dithiothreitol, Thiol-disulfide exchange


Mefenamic acid, (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug (NSAID) is metabolized into the chemically-reactive, MFA-1-O-acyl-glucuronide (MFA-1-O-G), MFA-acyl-adenylate (MFA-AMP), and the MFA-S-acyl-CoA (MFA-CoA), all of which are electrophilic and capable of acylating nucleophilic sites on biomolecules. In this study, we investigate the non-enzymatic ability of each MFA acyl-linked metabolite to transacylate amino and thiol functional groups on the acceptor biomolecules glycine (Gly), taurine (Tau), glutathione (GSH), and N-acetylcysteine (NAC). In vitro incubations with each of the MFA acyl-linked metabolites (1 μM) in buffer under physiological conditions with Gly, Tau, GSH, or NAC (10 mM) revealed that MFA-CoA was 11.5- and 19.5-fold more reactive than MFA-AMP towards the acylation of cysteine-sulfhydryl groups of GSH and NAC, respectively. However, MFA-AMP was more reactive towards both Gly and Tau, 17.5-fold more reactive towards the N-acyl-amidation of taurine than its corresponding CoA thioester, while MFA-CoA displayed little reactivity towards glycine. Additionally, MFA-GSH was 5.6- and 108-fold more reactive towards NAC than MFA-CoA and MFA-AMP, respectively. In comparison to MFA-AMP and MFA-CoA, MFA-1-O-G was not significantly reactive towards all four bionucleophiles. MFA-AMP, MFA-CoA, MFA-1-O-G, MFA-GSH, and MFA-Tau were also detected in rat in vitro hepatocyte MFA (100 μM) incubations while MFA-Gly was not. These results demonstrate that MFA-AMP selectively reacts nonenzymatically with the amino functional groups of glycine and lysine, MFA-CoA selectively reacts nonenzymatically with the thiol functional groups of GSH and NAC, and MFA-GSH reacts nonenzymatically with the thiol functional group of GSH, all of which may potentially elicit an idiosyncratic toxicity in vivo.

Concepts: Amino acid, Amine, Functional group, Disulfide bond, Non-steroidal anti-inflammatory drug, Paracetamol, Ibuprofen, Carboxylic acid


The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and its dysregulation is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER) is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR), distinct signaling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulfide Isomerase (PDI) is an ER chaperone induced during ER stress that is responsible for the formation of disulfide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However, specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

Concepts: Proteins, Protein, Endoplasmic reticulum, Protein folding, Chaperone, Disulfide bond, Protein disulfide isomerase, John Wells


Cone snails are predatory creatures using venom as a weapon for prey capture and defense. Since this venom is neurotoxic, the venom gland is considered as an enormous collection of pharmacologically interesting compounds having a broad spectrum of targets. As such, cone snail peptides represent an interesting treasure for drug development. Here, we report five novel peptides isolated from the venom of Conus longurionis, Conus asiaticus and Conus australis. Lo6/7a and Lo6/7b were retrieved from C. longurionis and have a cysteine framework VI/VII. Lo6/7b has an exceptional amino acid sequence because no similar conopeptide has been described to date (similarity percentage <50%). A third peptide, Asi3a from C. asiaticus, has a typical framework III Cys arrangement, classifying the peptide in the M-superfamily. Asi14a, another peptide of C. asiaticus, belongs to framework XIV peptides and has a unique amino acid sequence. Finally, AusB is a novel conopeptide from C. australis. The peptide has only one disulfide bond, but is structurally very different as compared to other disulfide-poor peptides. The peptides were screened on nAChRs, NaV and KV channels depending on their cysteine framework and proposed classification. No targets could be attributed to the peptides, pointing to novel functionalities. Moreover, in the quest of identifying novel pharmacological targets, the peptides were tested for antagonistic activity against a broad panel of Gram-negative and Gram-positive bacteria, as well as two yeast strains.

Concepts: Protein, Protein structure, Amino acid, Disulfide bond, Glutathione, Cysteine, Conus, Conidae


Glutathione S-transferase pi 1 (GSTP1), is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased glutathione (GSH). This data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at PLs C7-C8 olefin, while PLs C2-C3 olefin was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the x-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Angstrom resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; No covalent bond formation between hPL and GSTP1 was observed. Mass spectrometric (MS) analysis of reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting hPL is not membrane permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL:GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Concepts: Cancer, Cell division, Chemical bond, Disulfide bond, Glutathione, Covalent bond, Electrophile, Pi bond


One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Concepts: Sperm, Spermatozoon, Disulfide bond, Glutathione, Cysteine, Thiol


Well-aligned CdS nanorod arrays (CdS NRs) with ∼100nm in diameter and ∼700nm in length were fabricated on FTO (fluorine-doped tin oxide) substrate by using glutathione as capping agents. The growth of CdS NRs was studied in details by exploring the roles of each active binding group in glutathione. The thiol group in glutathione plays an important role in forming a compact CdS nanocrystal film, upon which the nanorods grow subsequently via the synergetic effect of thiol and dicarboxyl groups in glutathione. The influence of surface passivation with glutathione on the photoelectrical property of CdS NRs was also tested. The results revealed that glutathione ligands encapsulated in the surfaces of CdS NRs act as insulating barriers between CdS NRs and solution, hindering charge transport. Hybrid photovoltaic cells of FTO/CdS NRs/P3HT (poly(3-hexylthiophene))/Au were then assembled. The performance of the photovoltaic devices was increased with increasing the length of the as-prepared CdS nanorods and further enhanced to the highest efficiency of 0.373% after the thermal sulfuration treatment.

Concepts: Zinc, Disulfide bond, Sulfur, Cadmium, Photovoltaics, Cysteine, Thiol, Cadmium sulfide


Biothiols, such as cysteine (Cys) and homocysteine (Hcy), play very crucial roles in biological systems. Abnormal levels of these biothiols are often associated with many types of diseases. Therefore, the detection of Cys (or Hcy) is of great importance. In this work, we have synthesized an excellent “OFF-ON” phosphorescent chemodosimeter 1 for sensing Cys and Hcy with high selectivity and naked-eye detection based on an Ir(III) complex containing a 2,4-dinitrobenzenesulfonyl (DNBS) group within its ligand. The “OFF-ON” phosphorescent response can be assigned to the electron-transfer process from Ir(III) center and C^N ligands to the DNBS group as the strong electron-acceptor, which can quench the phosphorescence of probe 1 completely. The DNBS group can be cleaved by thiols of Cys or Hcy, and both the (3) MLCT and (3) LC states are responsible for the excited-state properties of the reaction product of probe 1 and Cys (or Hcy). Thus, the phosphorescence is switched on. Based on these results, a general principle for designing “OFF-ON” phosphorescent chemodosimeters based on heavy-metal complexes has been provided. Importantly, utilizing the long emission-lifetime of phosphorescence signal, the time-resolved luminescent assay of 1 in sensing Cys was realized successfully, which can eliminate the interference from the short-lived background fluorescence and improve the signal-to-noise ratio. As far as we know, this is the first report about the time-resolved luminescent detection of biothiols. Finally, probe 1 has been used successfully for bioimaging the changes of Cys/Hcy concentration in living cells.

Concepts: Amino acid, Disulfide bond, Luminescence, Cysteine, Phosphorescence, Thiol, Methionine, Photoluminescence


The bonding characteristics in cysteine-gold cluster complexes represented by thiolate (Au(n)·Cys(S) (n = 1, 3, 5, 7)) and thiol (Au(n)·Cys(SH) (n = 2, 4, 6, 8)) is investigated by density functional theory with 6-31G(d,p) and Lanl2DZ hybrid basis sets. The complexes exhibit very different bonding characteristic between these two forms. In the Au(n)·Cys(S) complexes, the charge transfers from gold clusters to sulfur atoms. The number of S-Au bonds in the Au(n)·Cys(S) complexes evolves from one to two when n is greater than three. For n equals three, i.e. Au(3)·Cys(S), its ground state only has one S-Au bond. While the only S-Au bond in Au(1)·Cys(S) is mainly covalent, the nature of the S-Au bond in other thiolates is featured with the combination of covalent and donor-acceptor interactions. In particular, one stable isomer of Au(3)·Cys(S) with two S-Au bonds, which is 2 kcal mol(-1) higher in energy than the corresponding ground state, consists of one covalent and one donor-acceptor S-Au bond explicitly. Moreover, the localized three center two electron bonds are formed within the Au clusters, which facilitates the formation of the two S-Au bonds in Au(5)·Cys(S) and Au(7)·Cys(S) complexes. In the Au(n)·Cys(SH) complexes, the donor-acceptor interaction prevails in the Au-SH bond by transferring lone pair electrons from the sulfur atom to the adjacent gold atom. Interestingly, the orbital with much more 6s-component in Au(4)·Cys(SH) enhances the donor-acceptor bonding character, thus yields the strongest bonding among all the Au(n)·Cys(SH) complexes studied in this paper. In general, the bonding strength between gold clusters and cysteine is positively correlated with the S-Au overlap-weighted bond order, but negatively correlated with the S-Au bond length. Lastly, the covalent and donor-acceptor S-Au bond strength is computed to be 48 and 18 kcal mol(-1), respectively.

Concepts: Electron, Atom, Chemical bond, Disulfide bond, Quantum chemistry, Bond order, Thiol, Bond length