Comparative (13)C and (31)P NMR studies of the ligand exchange reactions of auranofin with ergothionine, imidazolidine-2-thione and diazinane-2-thione.

The interaction of auranofin (Et(3)PAuSATg) with ergothionine (ErS), imidazolidine-2-thione (Imt) and diazinane-2-thione (Diaz) has been studied using (13)C and (31)P NMR spectroscopy. It is observed that these thiones are able to replace both Et(3)P and SATg(-) ligands simultaneously from gold(I) in auranofin forming >C [double bond] S-Au-SATg and [Et(3)P-Au-S [double bond] C<](+) type complexes. The displaced SATg(-) is oxidized to its disulfide (SATg)(2). However, some of the displaced Et(3)P is oxidized to Et(3)PO while the remaining reacts with thiones to form Et(3)P-S [double bond] C< species characterized by delta (31)P NMR of 1.0-1.5 ppm. The Et(3)PO resonance appeared in the 31P NMR spectrum, after 10 days of the addition of ErS, after 19 days of the addition of Imt and after 6 days of the addition of Diaz, to auranofin solution showing that the thiones react with auranofin very slowly.     

(13)C, (31)P and (15)N NMR studies of the ligand exchange reactions of auranofin and chloro(triethylphosphine)gold(I) with thiourea.

The interaction of thiourea (Tu) with auranofin (Et(3)PAuSATg) and its analogue, Et(3)PAuCl has been studied using (13)C, (31)P and (15)N NMR spectroscopy. It is observed that Tu is able to replace both the ligands, Et(3)P and SATg(-) simultaneously from gold(I) in auranofin, forming [Et(3)P-Au-Tu](+) and Tu-Au-SATg complexes. However, no separate resonances for these species were observed either due to their rapid exchange with auranofin and thus giving only the average resonances or because the chemical shifts of either two species are same so that they cannot be resolved. The displaced SATg(-) is oxidized to its disulfide, (SATg)(2). However, some of the displaced Et(3)P is oxidized to Et(3)PO while the remaining reacts with Tu to form Et(3)P-Tu species, characterized by delta 31P of 1.0 ppm, assigned after an independent reaction between Et(3)P and Tu. In an experiment using a 0.05 M solution of auranofin, the Et(3)PO resonance appeared in auranofin spectrum after 4 days of addition of 1.0 equivalent of Tu, showing that the reaction is slow. A resonance for free Et(3)P is also detected in 31P NMR on the addition of CN(-). It is also observed that Tu reacts with Et(3)PAuCl to form [Et(3)P-Au-Tu](+) via displacement of Cl(-), consistent with an upfield shift of 6.2 ppm in >C [double bond] S resonance of Tu in (13)C NMR. In (15)N NMR, a smaller downfield, instead of an upfield shift, in NH(2) resonance of Tu on its addition to auranofin and Et(3)PAuCl indicates that it is not binding to gold(I) through nitrogen.     

The reactions of hemopexin and liver fatty acid-binding protein (L-FABP) with aurothiomalate

We investigated whether gold(I) binds to hemopexin, Hx, a serum heme carrier with a molecular weight similar to that of serum albumin, or to L-FABP, a liver cytosolic heme- and fatty acid-binding protein whose molecular weight is similar to that of metallothionein. These proteins and, for comparison, similar concentrations of bovine albumin were incubated with sodium aurothiomalate and then fractionated by gel exclusion chromatography. At 16 μM of Hx and gold the ratio of gold bound to Hx was 0.30 ± 0.04. The corresponding ratio for albumin was 0.98 ± 0.04 per mercaptalbumin. Considering the much lower serum levels of Hx compared to albumin it is unlikely that Hx plays a role in serum gold binding and transport. L-FABP also binds gold: at 52 μM L-FABP and 108 μM gold, the gold to protein ratio was 0.55 ± 0.05. The corresponding ratio for albumin under identical conditions was 1.18 ± 0.08 per mercaptalbumin. L-FABP failed to bind zinc or cadmium, two other metals bound by metallothionein.     

Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin

The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (approximately 9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.     

Further studies on the resolution of human mercapt- and nonmercaptalbumin and on human serum albumin in the elderly by high-performance liquid chromatography

High-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on Asahipak GS-520 showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the secondary one to nonmercaptalbumin (HNA). HPLC analysis of HSA on Asahipak ES-520 N showed three peaks, the principal component corresponding to HMA, the secondary one to HNA having mixed disulfide with cysteine or glutathione and the tertiary one to HNA oxidized higher than mixed disulfide. Two kinds of rapid HPLC for the resolution of HSA into HMA and HNA were developed by the present authors. Using these HPLC, the present authors found a significant decrease in the fraction of HMA in the elderly.     

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

Simple and sensitive high-performance liquid chromatographic method for the investigation of dynamic changes in the redox state of rat serum albumin

Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MA(r)) and rat non-mercaptalbumin (NA(r)), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MA(r) fraction in control and exercise groups were 76.2+/-1.8 and 69.0+/-3.5%, respectively. The MA(r) in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress.     

A gold(I) complex with a vitamin K3 derivative: characterization and antitumoral activity

Reaction of the vitamin K(3) derivative menadione sodium bisulfite thiosemicarbazone (NaK(3)TSC) with chloro(triethylphosphine)gold(I) afforded the complex [AuPEt(3)(K(3)TSC)]. This compound consists of discrete molecules in which the metal is almost linearly coordinated to P and S. Preliminary in vitro screening showed significant anti-cancer activity, notably against the cisplatin-resistant cell line A2780cis.     

Aurothionein formation from Zn,Cd-thionein and Et3PAuCl,but not Et3PAuSATg (auranofin)

Et₃PAuCl reacts in vitro with horse-kidney zinc, cadmium-metallothionein (Zn, CdTh) to displace zinc, but not cadmium. The ratio of gold-bound to zinc displaced is larger than that observed for gold sodium thiomalate (AuSTm), suggesting a different mode of binding. Et₃PAuSATg (Auranofin: 2,3,4,6- tetra-O-acetyl-1-thio-β-D-glucopyranosato(triethyl- phosphine)gold(I)), a new antiarthritic compound, does not react with Zn, CdTh. The significance of the nonreaction of Et₃PAuSATg, in contrast to Et₃PAuCl and AuSTm (previously studied), for the in vivo pharmacology of gold is discussed.     

Inhibition of cathepsin B by Au(I) complexes: a kinetic and computational study

Gold(I) compounds have been used in the treatment of rheumatoid arthritis for over 80 years, but the biological targets and the structure–activity relationships of these drugs are not well understood. Of particular interest is the molecular mechanism behind the antiarthritic activity of the orally available drug triethylphosphine(2,3,4,6-tetra-O-acetyl-β-1-d-thiopyranosato-S) gold(I) (auranofin, Ridaura). The cathepsin family of lysosomal, cysteine-dependent enzymes is an attractive biological target of Au(I) and is inhibited by auranofin and auranofin analogs with reasonable potency. Here we employ a combination of experimental and computational investigations into the effect of changes in the phosphine ligand of auranofin on its in vitro inhibition of cathepsin B. Sequential replacement of the ethyl substituents of triethylphosphine by phenyl groups leads to increasing potency in the resultant Au(I) complexes, due in large part to favorable interactions of the more sterically bulky Au(I)–PR₃ fragments with the enzyme active site.     

Peroxidase-catalyzed-3-(glutathion-S-yl)-p,p'-biphenol formation

Oxidation of p,p'-biphenol with horseradish peroxidase (HRP)-hydrogen peroxide in the presence of bovine serum albumin or with bone marrow cell homogenate-hydrogen peroxide resulted in the formation of reactive products that conjugate with protein. Glutathione prevented the protein binding. Glutathione readily reacted with p,p'-biphenoquinone, the principal oxidation product of p,p'-biphenol in the HRP-hydrogen peroxide system and resulted in the formation of several glutathione conjugates, p,p'-biphenol and small amounts of oxidized glutathione. The major glutathione conjugate was identified as 3-(glutathion-S-yl)-p,p'-biphenol by high field nuclear magnetic resonance and fast atom bombardment mass spectrometry. The same conjugate was formed in the bone marrow homogenate-hydrogen peroxide system. p,p'-Biphenoquinone reduction by glutathione to p,p'-biphenol without glutathione oxidation was explained by the rapid reduction of p,p'-biphenoquinone by 3-(glutathion-S-yl)-p,p'-biphenol.     

Interaction of Et2SnCl2 with 5'-IMP and 5'-GMP

The interactions of Et2SnCl2 with 5'-IMP and 5'-GMP have been studied in aqueous solutions by 1H- and 31P-NMR spectroscopy as a function of pH. At low pH values (< 4.0) Sn(IV) interacts with the pyrophosphate oxygens of these nucleotides. At intermediate pH values (4-9.5) no interaction of the metal with the nucleotides take place, while at pH > 9.5 the sugar O'2 and O'3 atoms are the preferred coordination sites. In addition, the solid adducts obtained from aqueous solutions at pH = 3-4 of the above interactions correspond to formulae; (Et2Sn)2(5'-IMP)2(H2O) and (Et2Sn)3(5'-GMP)2(OH)2(H2O)2 as their elemental analysis show. IR spectra and solid state 13C, 31P-NMR spectra 119Sn M?ssbauer and solution 119Sn-NMR spectra once more confirm the pyrophosphate involvement in bonding with Sn(IV) in oligomeric or polymeric structures and trigonal bipyramidal or octahedral geometries.     

Yeast glutathione reductase. Studies of the kinetics and stability of the enzyme as a function of pH and salt concentration.

1. The pH dependencies of the apparent Michaelis constant for oxidized glutathione and the apparent turnover number of yeast glutathione reductase (EC 1.6.4.2) have been determined at a fixed concentration of 0.1 mM NADPH in the range pH 4.5--8.0. Between pH 5.5 and 7.6, both of these parameters are relatively constant. The principal effect of low pH on the kinetics of the enzyme-catalyzed reaction is the observation of a pH-dependent substrate inhibition by oxidized glutathione at pH less than or equal 7, which is shown to correlate with the binding of oxidized glutathione to the oxidized form of the enzyme. 2. The catalytic activity of yeast glutathione reductase at pH 5.5 is affected by the sodium acetate buffer concentration. The stability of the oxidized and reduced forms of the enzyme at pH 5.5 and 25 degrees C in the absence of bovine serum albumin was studied as a function of sodium acetate concentration. The results show that activation of the catalytic activity of the enzyme at low sodium acetate concentration correlates with an effect of sodium acetate on a reduced form of the enzyme. In contrast, inhibition of the catalytic activity of the enzyme at high sodium acetate concentration correlates with an effect of sodium acetate on the oxidized form of the enzyme.     

The 13C NMR study of the binding of gold(I) thiomalate with ergothionine in aqueous solution.

The interaction of gold(I) thiomalate (Autm) (Myocrysine) with ergothionine (ErSH) has been studied in aqueous solution at pH 7.4. It was found that ErSH forms a ternary complex of the type ErS-Au-tm at a 1:1 mole ratio; unlike other thiols (e.g., cysteine and glutathione) it does not eject thiomalate (Htm) as a free ligand. However, in the presence of glutathione (GtSH), both ligands, ErSH as well as Htm, from the ErS-Au-tm complex were freed. The 13C NMR chemical shifts of C-2 resonance of ergothionine in the presence of Autm shifts greater than imidazolidine-2-thione (Imt) and 1,3-Diazinane-2-thione (Diaz), indicating the stronger complexation of ErS-Au-tm compared to Imt-Au-tm and Diaz-Au-tm.     

13C nmr studies of aurothioglucose: Ligand exchange and redox disproportionation reactions

¹³C nmr studies of gold thioglucose, AuSTg, and solutions containing added β-1-D-thioglucose, TgSH, have been conducted at PD 7.4 and interpreted in terms of complexation and ligand exchange reactions that are consistent with the known preference of gold(I) for linear two-coordinate structures. The upper limit of the half-life for ligand exchange between 0.25 M Au(STg)₂^? and TgSH at pD 7.4 is 2.2 msec. The ¹³C nmr spectra of various thioglucose derivatives have been assigned. A novel oxidation reduction reaction was discovered that leads to the formation of metallic gold and a product tentatively identified as the sulfinic acid derivative of thioglucose. The presence of sulfinic acid in AuSTg was indicated by the infrared absorption at 1050 cm^?1. The same product was formed by slow hydrolysis of thioglucose disulfide. A mechanism for the formation of the sulfinic acid derivative from AuSTg is proposed.     

Bis(L-cysteinato)gold(I): chemical characterization and identification in renal cortical cytoplasm.

L-Cysteinatogold(I) was prepared by the reaction of L-cysteine with KAuBr4 in acidic media and its solubility determined from pH 4 to 10. The solubility at pH 7.4 and 37 degrees C is 1 microM. In the presence of excess cysteine, the solubility increases because of formation of bis(L-cysteinato)gold(I). The equilibrium-constant for formation of the bis complex is 2.1 +/- 0.4 X 10(-3), which at pH 7.4 CORRESPONDs to an apparant formation constant of 4.4 X10(4). The formation of the bis adduct was confirmed by chromatographic separation of the products of the reaction between [35S]-L-cysteine and Na2AuTM. This complex elutes with Kav = 1.15 which allows it to be distinguished from other gold thiolates that might form in vivo. The bis(cysteinato)gold(I) complex is shown to be present in kidney cytosol isolated from rats given Na2AuTM in vivo. When additional cysteine is added to the cytosol in vitro, the peak at 1.15 is increased, but if glutathione is added, the low molecular weight gold elutes at Kav = 1.00, which is taken as evidence for the existence of bis(cysteinato)gold(I) in the cytosol preparation. The amount of gold present as bis(cysteinato)gold(I) after 4 different dose schedules has been measured and found to increase with the total cytosol gold concentration. L-Cysteinatogold(I) does not dissolve in the presence of bovine serum albumin to form an adduct.     

Histochemical localization of oxidized glutathione-catalysing enzymes in human term placenta

Summary This paper reports a new cytochemical affinity technique for detecting oxidized-glutathione-binding enzymes by light microscopy and for carrying out fine structural analyses by means of oxidized glutathione labelled with colloidal gold. Albumin-colloidal gold particles were prepared. Oxidized glutathione, added to the solution, replaced albumin on the surface of the gold, thus forming a new histochemical reagent. In human placenta, oxidized-glutathione-catalysing activity was detected on the brush border of the placental villi, over the foldings of endothelial membranes, and in the ground substance of connective tissue in the villous core. Every process of the cell membrane demonstrated enhanced oxidized-glutathione-catalysing activity. This new reagent is a unique example of a low-molecular-weight compound labelled with colloidal gold, and it permits direct measurement of oxidized-glutathione-binding activity in tissue sections.     

Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture

The nonionic 2'-O-methyribooligonucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, is complementary to the...ApCpC...sequence found in the amino acid accepting stem of most tRNAs and the anticodon region of tRNAgly and to the threonine codon of mRNA. Gmp(Et)Gmp(EtU forms hydrogen-bonded complexes with the amino acid accepting stem of tRNApheyeast and unfractionated tRNA Escherichia coli under physiological salt conditions at 37 degrees C as determined by equilibrium dialysis. The extent of phenylalanine aminoacylation of tRNApheE.coli is inhibited 39% by Gmp(Et)Gmp(Et)U at 37 degrees C in solution. The triester is resistant to hydrolysis by serum nucleases and cell lysates. The triester is readily taken up by transformed Syrian hamster fibroblasts growing in monolayer. Within the cell, the triester is deethylated to give the trinucleotide species Gmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU and is also hydrolyzed to dimeric and monomeric units. Treatment of transformed fibroblasts in monolayer with 25 micronM Gmp(Et)Gmp(Et)U results in a 40% inhibition of cellular protein synthesis with a concurrent slight increase in cellular RNA synthesis during the first 4 h. After 4 h, the rate of cellular protein synthesis begins to recover while RNA synthesis returns to that of the control. Our biochemical studies suggest that inhibition of cellular protein synthesis might be expected if Gmp(Et)Gmp(Et)UGmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU, which have been taken up by or formed within the cell, physically bind to tRNA and mRNA and inhibit the function of these nucleic acids. The reversible inhibition of protein synthesis may be a consequence of further degradation of the trinucleotide species within the cell as well as to an increase in supply of RNA molecules involved in protein synthesis. The growth of the transformed fibroblasts is inhibited during the first 24 h of incubation with 25 micronM Gmp(Et)Gmp(Et)U after which growth proceeds at a normal rate. In cloning experiments, the number and size of colonies formed by the transformed fibroblasts after 5 days exposure to 25 micronM triester is decreased by 50% relative to untreated controls. The temporary inhibition of cell growth may reflect the transitory inhibition of cellular protein synthesis caused by the triester.     

Interaction of Gold(I) with the Active Site of Selenium-Glutathione Peroxidase

Gold(I) thioglucose in the presence of excess glutathione (GSH) leads to strong and reversible inhibition of selenium-glutathione peroxidase (EC 1.11.19) around neutral pH. Binding at equilibrium and competition studies demonstrated that the most reduced form of the active site selenocysteine is the only binding site for gold(I) Steady-state kinetics indicate that gold(I) forms a dead-end complex with glutathione peroxidase in competition with the reduction of hydroperoxide. The apparent K₁ is 2 3 μM at pH 7.6,37°C and 1 mM GSH. Theoretical models of inhibition were assessed by the use of linear least-squares fitting to a generalized integrated rate equation. The results are consistent with trapping of gold(I) at the active site in the form of a mixed bidentate selenolato-thiolate complex involving GSH and the active site selenocysteine. The kinetics of inhibition imply that the resting form of glutathione peroxidase in the presence of excess GSH is also within the enzyme cycle. This rules out the existence of selenium(+lV) species in the redox cycle of the active site when t-butylhydroperoxide is used as a substrate. Electronic properties of selenium and gold as well as a large relief of inhibition by selenocysteine suggest that a very stable interaction should be obtained between Se(-II) and gold(I) through covalent bonding. These results suggest that glutathione peroxidase could be a target of gold drugs used in the treatment of rheumatoid arthritis.     

Glutathionato-S-Gold(III) complexes formed as intermediates in the reduction of auricyanide by glutathione

The reduction of auricyanide ([Au(CN)(4)](-), a potential gold(III) metabolite of antiarthritic gold(I) compounds), by glutathione (G(-)SH, an anionic biological reductant) proceeds through two intermediates (I(230) and I(290)) which have previously been identified by their UV-vis spectra, but not isolated. Negative-ion electrospray ionization-mass spectroscopy (ESI-MS) has unambiguously identified them as [Au(CN)(3)(SG)](2-) and [Au(CN)(2)(SG)(2)](3-), respectively, and allowed their formation and decay to be monitored. The spectra also confirm that the products are aurocyanide ([Au(CN)(2)](-), a known metabolite of chrysotherapy agents) and oxidized glutathione (GSSG(2-)). The reactions are dependent on the presence or absence of buffering agents and the pH of the reaction media. The reaction can be driven to the first intermediate by using an excess of auricyanide or by running the reaction at low pH which prevents further reaction. At neutral pH and/or with excess of glutathione present, the reaction proceeds to the second intermediate, which is then reduced to aurocyanide. The monoanions, [Au(CN)(3)(SGH)](-) at m/z=581.2 and [Au(CN)(2)(SGH)(2)](-) at m/z=861.5 generate more intense signals than their respective dianions, [Au(CN)(3)(SG)](2-) at m/2=290.2 and [Au(CN)(2)(SG)(SGH)](2-)m/2=430.9, respectively, whereas the trianion [Au(CN)(2)(SG)(2)](3-) (m/3=281.2) was not observed. These studies demonstrate the value of ESI-MS methods for characterizing reactions of metallopharmaceuticals under biomimetic conditions and suggest that they will be useful for other systems which give strong ESI-MS signals.