Diaryl disulfides and thiosulfonates as combretastatin A-4 analogues: Synthesis, cytotoxicity and antitubulin activity.

Diaryl disulfides and diaryl thiosulfonates were synthesized with the two phenyl rings of all compounds bearing identical halide substituents. Because of structural similarity to the potent antimitotic natural product combretastatin A-4 (CA-4), the compounds were examined for inhibition of tubulin polymerization, and the thiosulfonates were more active than the disulfides. The nine thiosulfonates had IC50 values ranging from 1.2 to 9.1 µM, as compared with 1.3 µM obtained with CA-4. The compounds thus ranged from equipotent with CA-4 to 7-fold less active. The nine disulfides had IC50 values ranging from 1.2 to 5.1 µM, as compared with 0.54 µM obtained with CA-4. The compounds thus ranged from less than half as active as CA-4 to over 9-fold less active. The most active members of each group, 2 g and 3c, in the assembly assay were modeled into the colchicine site. Compound 3c had significant hydrophobic interactions with ß-tubulin residues CYS 241 and ALA 250, and its thiosulfonate bridge made a hydrogen bond with ß-tubulin residue ASN 258. Compound 2 g had hydrophobic interactions with ß-tubulin residues ALA 250, CYS 241 and ALA 254, but there was no significant interaction of the disulfide bridge with tubulin.

Cost Effective Use of a Thiosulfinate-Enriched Allium sativum Extract in Combination with Chemotherapy in Colon Cancer.

In this work, we sought to investigate the effects of a thiosulfinate-enriched garlic extract, co-administered with 5-fluorouracil (5-FU) or oxaliplatin chemotherapy, on the viability of colon cancer cells (Caco-2 and HT-29). We also addressed the economic feasibility of a new combined treatment of this thiosulfinate-enriched garlic extract, with oxaliplatin that could reduce the dosage and costs of a monotherapy. The thiosulfinate-enriched garlic extract not only enhanced the impact of 5-FU and oxaliplatin (500 µM) in decreasing Caco-2 and HT-29 viability, but also showed a higher effect than standard 5-FU and oxaliplatin chemotherapy as anti-cancer agents. These results provided evidences for the combination of lyophilized garlic extract and 5-FU or oxaliplatin as a novel chemotherapy regimen in colon cancer cells that may also reduce the clinical therapy costs.

A Comparison of the Antibacterial and Antifungal Activities of Thiosulfinate Analogues of Allicin.

Allicin (diallylthiosulfinate) is a defence molecule from garlic (Allium sativum L.) with broad antimicrobial activities in the low µM range against Gram-positive and -negative bacteria, including antibiotic resistant strains, and fungi. Allicin reacts with thiol groups and can inactivate essential enzymes. However, allicin is unstable at room temperature and antimicrobial activity is lost within minutes upon heating to >80 °C. Allicin's antimicrobial activity is due to the thiosulfinate group, so we synthesized a series of allicin analogues and tested their antimicrobial properties and thermal stability. Dimethyl-, diethyl-, diallyl-, dipropyl- and dibenzyl-thiosulfinates were synthesized and tested in vitro against bacteria and the model fungus Saccharomyces cerevisiae, human and plant cells in culture and Arabidopsis root growth. The more volatile compounds showed significant antimicrobial properties via the gas phase. A chemogenetic screen with selected yeast mutants showed that the mode of action of the analogues was similar to that of allicin and that the glutathione pool and glutathione metabolism were of central importance for resistance against them. Thiosulfinates differed in their effectivity against specific organisms and some were thermally more stable than allicin. These analogues could be suitable for applications in medicine and agriculture either singly or in combination with other antimicrobials.

ROSics: chemistry and proteomics of cysteine modifications in redox biology.

Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels.

Cysteine reacts to form blue-green pigments with thiosulfinates obtained from garlic (Allium sativum L.).

Cysteine was found to form pigments with garlic thiosulfinates in this investigation, in contrast to previous reports. Pigments were formed only when the molar concentration ratios of cysteine to total thiosulfinates were smaller than 2:1. Cysteine does not form pigments with thiosulfinates in the same manner as other pigment-forming amino compounds because it has a sulfhydryl (SH) group. A colour reaction of cysteine with thiosulfinates is proposed where colourless disulphide-type S-alk(en)yl mercaptocysteines (SAMCs) are formed first by the SH-involved reaction between cysteine and thiosulfinates, and then SAMCs react with residual thiosulfinates to form pigments. When the cysteine to total thiosulfinate molar concentration ratio was 2:1 or greater, total thiosulfinates were consumed to form SAMCs without leaving any thiosulfinates remaining available for the following colour reactions.

In vitro-in vivo study on the effects of plant compounds on rumen fermentation, microbial abundances and methane emissions in goats.

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P≥ 0.366). Total volatile fatty-acid (VFA) concentration decreased (P≤ 0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P≤ 0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P≤ 0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P≤ 0.004) by CAR and PTS. The addition of all compounds linearly decreased (P≤ 0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet-dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P≤ 0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.

The topology of the cardiac Na⁺/Ca²âº exchanger, NCX1.

The topology of the plasma membrane Na(+)/Ca(2+) exchanger of cardiac muscle, NCX1, is uncertain. Biochemical analyses have indicated the presence of 9 transmembrane segments (TMSs) whereas the recent crystal structure of a prokaryotic homologue has 10 TMSs. The discrepancy is towards the C-terminus of the proteins where the prokaryotic homologue has an additional TMS8. To resolve this apparent disagreement, we re-assessed the topology of the C-terminal TMSs of NCX1. We examined the ability of internal or external cysteine residues in the N-terminal portion of NCX1 to crosslink with cysteine residues, of uncertain orientation, in the C-terminal portion of the protein. The results strongly support a model of NCX1 with 10 TMSs as found in the prokaryotic homologue.

Cloning and expression of γ-glutamyl transpeptidase and its relationship to greening in crushed garlic (Allium sativum) cloves.

BACKGROUND: Garlic greening occurs when garlic cloves are stored at low temperature, increasing 1-propenyl cysteine sulfoxide, which is induced by γ-glutamyl transpeptidase (GGT) activity. Although the metabolism of the γ-glutamyl peptide is important for the biosynthesis of green pigments in crushed garlic cloves, garlic GGT is poorly characterised. RESULTS: For the analysis of GGT at the gene level, the garlic GGT sequence was partially cloned using an onion GGT sequence. The relationship between garlic greening and related gene expressions, depending on storage condition, was investigated using reverse transcription polymerase chain reaction for garlic GGT and alliinase. Three storage conditions were set: A, storage at a constant temperature of 20 °C; B, storage at 20 °C for 3 months and then transfer to 0 °C for an additional 3 months; C, storage at 0 °C for 3 months and then transfer to 20 °C for an additional 3 months. GGT expression increased under storage condition B and decreased under storage condition C. However, alliinase expression was not affected by storage condition. CONCLUSION: Greening in crushed garlic cloves increases with increasing GGT expression at low temperature, while alliinase expression is not affected.

Synthetic water-soluble phenolic antioxidant regulates l-arginine metabolism in macrophages: a possible role of Nrf2/ARE.

Synthetic water-soluble phenolic antioxidant TS-13 exhibits pronounced anti-inflammatory properties in vivo and induces intracellular signal system Nrf2/ARE. At concentrations 150-1000 microM it inhibits nitric oxide (NO) production in mouse peritoneal macrophages. However, this compound at low concentrations (1-100 microM) paradoxically increases NO production and decreases activity of arginase. These results are indicative of an ambiguous role of NO and its metabolites in the mechanism of development of inflammatory reaction.

[Variability of the exogenic antioxidant effect on survival: modeling in Drosophila lines with different lifespan and l(2)gl-tumor suppressor dosage].

Different exogenic antioxidants and geroprotectors are used to decrease age abnormalities and enhance the human life span. However, the antioxidant effect on lifespan is variable and requires detailed analysis. In the present report, we modeled in Drosophila the peculiar character of action of various doses of a new phenol antioxidant TC-13. We studied the TC-13 effect on aging of two Drosophila lines with genetically determined contrast lifespan dynamics. In addition, we tested the TC-13 antioxidant influence on the background of heterozygozity on the loss-of-function mutation of the l(2)gl tumor suppressor. The differing effect of TS-13 on LS, the character of which depends on the antioxidant dosage, genotype of line, and sex of Drosophila, was found. TS-13 in the concentration 0.2% did not affect the lifespan in all studied lines and decreased survival, whereas the antioxidant in a concentration of 1% positively affected the lifespan in both males and females of long-living lines. The antioxidant effect on animal lines with a smaller dose of tumor suppressor l(2)gl resulted in a decrease of the lifespan.

Cysteine and glutathione mixed-disulfide conjugates of thiosulfinates: chemical synthesis and biological activities.

The chemical syntheses of cysteine (CYS) and glutathione (GSH) mixed -disulfide conjugates (CySSR, GSSR, respectively) of mercapto residues representing most of the R groups of thiosulfinates (R = methyl, ethyl, propyl, and allyl) are described. Gram-scale conjugates were prepared as >98% pure preparations, with 80% reaction yield for each of the two seminal synthesis steps, with structures confirmed by (1)H NMR and high-resolution MS analyses. These conjugates are derivatives of thiosulfinates that may be evolved in processed foods, in the digestive tract, and through in vivo metabolism. The prepared conjugates were found to be able to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7), indicating they have potential cancer preventive and anti-inflammatory activities. Among the prepared conjugates, the allyl conjugates of CYS and GSH, S-allylmercaptocysteine (CySSA) and S-allylmercaptoglutathione (GSSA), showed the most potent activity regarding QR induction and NO production inhibition. The conjugates with saturated R groups were also active and conferred biological activity as cystine and oxidized glutathione exhibited no effects in these cellular assays.

Reactive sulfur species: kinetics and mechanism of the equilibrium between cysteine sulfenyl thiocyanate and cysteine thiosulfinate ester in acidic aqueous solution.

The kinetics and mechanism of the hydrolysis of cysteine sulfenyl thiocyanate (CySSCN) to give cysteine thiosulfinate ester (CyS(=O)SCy) have been investigated between pH 0 and 4. The reaction is reversible. The hydrolysis of CySSCN is second-order in [CySSCN] and inverse first-order in [H+] and [SCN-]. The following mechanism is proposed for the hydrolysis of CySSCN (where the charge depends upon the pH): CySSCN0/+ + H2O <==>CySOH0/+ + SCN- + H+, CySOH0/+ + CySSCN0/+ --> CyS(=O)SCy0/+/2+ + SCN- + H+; k1 = 3.36 +/- 0.01 x 10-3 s-1, K1k2 = 0.13 +/- 0.05 Ms-1 (which yields k2/k-1 = 39 M). The observed rate law rules out alternative mechanisms for 1 0.4 M). The following mechanism is proposed: CyS(=O)SCy2+ + H+ <==> CyS(OH)=SCy3+, Ka; CyS(OH)SCy3+ + SCN- --> CySOH+ + CySSCN+, k-2 = 0.239 +/- 0.007 M-2s-1/Ka M-1. Since cysteine sulfenic acids are known to play an important function in many enzymes, and SCN- exists in abundance in physiologic fluids, we discuss the possible role of sulfenyl thiocyanates in vivo.

New sulfurated derivatives of valproic acid with enhanced histone deacetylase inhibitory activity.

One dithiolthione and two new methanethiosulfonate derivatives of valproic acid (VPA) were synthesized and tested in vitro as histone deacetylase (HDAC) inhibitors. The new molecules, as well as their sulfurated moieties, exhibited a much stronger inhibition of HDAC enzymatic and antiproliferative activities and histone hyperacetylation than VPA. ACS 2 is the most interesting compound among the new VPA derivatives and its sulfurated moiety, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, also known to be a metabolite of anethole trithione, seems to contribute significantly to its activity. This is the first time that HDAC inhibitory activity is described for dithiolethiones and thiosulfonates.

Reactive sulfur species: kinetics and mechanism of the hydrolysis of cysteine thiosulfinate ester.

The kinetics and mechanisms of the hydrolysis of cysteine thiosulfinate ester (CyS(O)SCy ( x- ), x = 0-2) have been investigated by stopped-flow spectrophotometry between pH 6 and pH 14. The rate-limiting reaction of hydroxide is observed for pH < 13. More complicated kinetics are observed above pH 13, where the hydrolysis of CyS(O)SCy (2-) can be fast relative to subsequent reactions. The eventual products of hydrolysis are a 1:1 molar ratio of cystine (CySSCy) and cysteine sulfinic acid (CySO 2H) under all reaction conditions. The rate of hydrolysis is dependent upon the proton state of CyS(O)SCy ( x- ). Furthermore, cysteine thiosulfonate ester (CyS(O) 2SCy) was observed as an intermediate during the hydrolysis of CyS(O)SCy ( x- ) at lower pH. CyS(O) 2SCy eventually hydrolyzes to give stoichiometric amounts of CySSCy and CySO 2H. However, CySO 2H is observed under some conditions for which hydrolysis of CyS(O) 2SCy is relatively slow, thus suggesting multiple hydrolysis pathways for CyS(O)SCy ( x- ). The mechanism up to the rate-limiting step is proposed to be as follows: CyS(O)SCy (0) = H (+) + CyS(O)SCy (-), p K a3 = 7.32; CyS(O)SCy (-) = H (+) + CyS(O)SCy (2-), p K a4 = 7.92; CyS(O)SCy (0) + OH (-) --> products, P 0 k 0 = (5.0 +/- 0.01) x 10 (3) M (-1) s (-1); CyS(O)SCy (-) + OH (-) --> products, P 1 k 1 = 60 +/- 18 M (-1) s (-1); and CyS(O)SCy (2-) + OH (-) --> products, P 2 k 2 = 0.36 +/- 0.01 M (-1) s (-1), where P x is a constant (1

Bruguiesulfurol, a new sulfur compound from Bruguiera gymnorrhiza.

A new cyclic 4-hydroxy-dithiosulfonate, bruguiesulfurol (1), as well as two known 4-hydroxydithiolane 1-oxides, brugierol (2) and isobrugierol (3) were isolated from the flowers of Bruguiera gymnorrhiza. With stably-transfected HepG2 cells, the three isolates activated antioxidant response element (ARE) luciferase activation with (EC(50)) values of 56.7, 3.7 and 1.8 microM, respectively. Compounds 2 and 3 also inhibited phorbol ester-induced NF-kappaB (nuclear factor-kappaB) luciferase activity with IC (50) values of 85.0 and 14.5 microM, respectively. In addition, compound 2 inhibited enzyme cyclooxygenase-2 (COX-2) activity with an IC(50) value of 6.1 microM. The structures of these isolates were determined by spectral data, and that of compound 1 was confirmed by X-ray crystallographic analysis.

Site-directed mutagenesis and use of bile acid-MTS conjugates to probe the role of cysteines in the human apical sodium-dependent bile acid transporter (SLC10A2).

The residues involved in substrate interaction of the human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) remain undefined. Biochemical modification of conserved cysteine residues has suggested their direct involvement in hASBT function. In the present study, we developed novel methanethiosulfonyl (MTS)-bile salt derivatives and describe their reactivity toward hASBT and its mutants. Endogenous Cys residues were subjected to Ala/Thr scanning mutagenesis and subsequent exposure to affinity inactivators. We show that C51A/T, C105A/T, C144A, and C255A/T are loss-of-function mutations. Additionally, C74A/T cell surface expression was abolished suggesting a role in protein folding and/or trafficking. C270A remained largely unaffected in the presence of 1.0 mM polar and charged MTS reagents (MTSEA, MTSES, and MTSET) and retained function similar to wt-hASBT control. However, in the presence of synthetic cholyl- and chenodeoxycholyl-MTS analogues, C270A displayed a significant decrease in K(T) and J(max). Our findings demonstrate that Cys270 is a highly accessible extracellular residue susceptible to thiol modification in its native form that remains largely unaffected upon mutation to Ala. Consequently, C270A provides an ideal scaffold for cysteine scanning mutagenesis studies. Furthermore, the substantial decrease in ligand affinity and maximal transport capacity of C270A suggest that C270 may potentially impact, although not critically, a putative substrate binding domain of hASBT. Overall, bile acid-MTS conjugates can serve as novel and powerful tools to probe the role of endogenous as well as engineered Cys residues and, ultimately, aid in defining their role in the bile acid binding region(s) of hASBT.

Glycosyl phenylthiosulfonates (glyco-PTS): novel reagents for glycoprotein synthesis.

Controlled site-selective glycosylation can be achieved by combining site-directed cysteine mutagenesis with chemical modification of the introduced thiol; a new class of more efficient chemoselective reagents, glycosyl phenylthiosulfonates, allow rapid glycosylations of representative simple thiols, peptides and proteins.

Probing the pore of ClC-0 by substituted cysteine accessibility method using methane thiosulfonate reagents.

ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.

Peptide synthesis using novel S-sulfocysteine derivatives.

New cysteine S-sulfonate derivatives, Boc-Cys(SO3Na)-ONa 2 and Fmoc-Cys(SO3Na)-ONa 3, were prepared and their utility for peptide synthesis examined. The Fmoc derivative 3 was used in the solid-phase peptide synthesis of Arg8-vasopressin 9 via the Bunte salt 7. Satisfactory S-sulfonate stability was observed when p-cresol scavenged the cleavage from the resin. The intermediate 7 was purified by ion-exchange chromatography prior to S-sulfonate cleavage with tributylphosphine.