C. Frank Shaw

Basal metallothionein in tumors: Widespread presence of apoprotein

A survey has been conducted of solid and ascites tumors from mice and solid tumors in rats for the presence of metallothionein or metallothionein-like protein. In most tumors, a positive identification was made on the basis of Sephadex G-75 and HPLC-DEAE chromatography followed by competitive radioimmunoassay for metallothionein. Apometallothionein was revealed in a number of tumors for the first time by comparing the Sephadex G-75 chromatographic profiles of Zn in native cytosol and Cd in cytosol incubated briefly with CdCl2 to saturate free binding sites on the protein before Sephadex G-75 chromatography. In two cases unsaturation of metallothionein was correlated with a lack of zinc in the ascites fluid which supplies the tumor with zinc.

Redox and ligand exchange reactions of potential gold(I) and gold(III)-cyanide metabolites under biomimetic conditions.

Biomimetic pathways for the oxidation of [Au(CN)(2)](-), a gold metabolite, and further cyanation of the gold(III) products to form Au(CN)(4)(-) were investigated using 13C NMR and UV-Visible spectroscopic methods. Hypochlorite ion, an oxidant released during the oxidative burst of immune cells, was employed. The reaction generates mixed dicyanoaurate(III) complexes, trans-[Au(CN)(2)X(2)](-), where X(-) represents equilibrating hydroxide and chloride ligands, and establishes the chemical feasibility of dicyanoaurate oxidation by OCl(-) to gold(III) species. This oxidation reaction suggests a new procedure for synthesis of H[Au(CN)(2)Cl(2)]. Reaction of trans-[Au(CN)(2)X(2)](-) (X(-)=Cl(-) and Br(-)) or [AuCl(4)](-) with HCN in aqueous solution at pH 7.4 leads directly to [Au(CN)(4)](-) without detection of the anticipated [Au(CN)(x)X(4-x)](-)intermediates, which is attributed to the cis- and trans-accelerating effects of the cyanides. The reduction of [Au(CN)(4)](-) by glutathione and other thiols is a complex, pH-dependent process that proceeds through two intermediates and ultimately generates [Au(CN)(2)](-). These studies provide further insight into the possible mechanisms of an immunogenically generated gold(I)/gold(III) redox cycle in vivo.

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein.

 − 1 s − 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn7-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M − 1 s − 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd2+ into the peptide. At pH 7.4 and 25 °C, Cd2+ has higher affinity for apometallothionein than for the apo-peptide.

Inhibition of Erythrocyte Selenium-Glutathione Peroxidase by Auranofin Analogues and Metabolites

The effect of gold ligation on the inhibition of bovine erythrocyte selenium-glutathione peroxidase (GSH-Px) was examined. The anti-arthritic drug auranofin [2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S)(triethylp hosphine) gold(I)] (Et3PAuSATg) and its analogue, Et3PAuCl, exhibited experimentally equivalent Ki values (11.6+/-0.8 and 10.8+/-0.5 microM, respectively), despite the greatly disparate affinities of their ligands for gold(I): 2,3,4,6-tetra-O-acetyl-1-thiolato-beta-D-glucopyranose (ATgS-) >> Cl-. This similarity reflects ligand exchange reactions that generate the glutathione complex Et3PAuSG from the excess glutathione (GSH, 1 mM) used in the assay. The Ki values for bis(glutathionato)gold(l) (Au(SG)2-) and gold(I) thioglucose (AuSTg) were also found to be equal (2.8+/-0.4 and 2.4+/-0.5 microM, respectively). This confirms the previous postulate of Chaudiere and Tappel (J Inorg Biochem 20: 313-325, 1984) that Au(SG)2- is generated from AuSTg in the presence of excess glutathione. Since auranofin metabolites accumulate in red blood cells, the inhibition of intracellular GSH-Px was examined by using intact erythrocytes. There was greater inhibition of the reaction when the cells were resuspended in isotonic buffer than in whole blood, because serum albumin in the latter competes for the auranofin and decreases the uptake by erythrocytes. After correction for the extent of gold uptake, the Ki values were determined to be the same as those observed for Au(SG)2- in the extracellular assay, indicating loss of both the Et3P and ATgS- ligands from auranofin. Thus, the inhibition of GSH-Px by gold complexes is dependent on their ligation, and the ultimate gold(I) compound that interacts with erythrocyte GSH-Px in intact red cells, Au(SG)2-, is radically different from the original auranofin molecule.

Seleno-auranofin (Et3PAuSe-tagl): synthesis, spectroscopic (EXAFS, 197Au Mössbauer, 31P, 1H, 13C, and 77Se NMR, ESI-MS) characterization, biological activity, and rapid serum albumin-induced triethylphosphine oxide generation.

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mössbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

Mechanism of Cadmium Ion Substitution in Mammalian Zinc Metallothionein and Metallothionein Alpha Domain: Kinetic and Structural Studies

Cellular metallothionein (MT) protects against Cd(2+) exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn(7)-MT-2 with Cd(2+) was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn(4)-alpha domain revealed that only the fastest step involved the Zn(3)-beta domain. Each step of the Zn(4)-alpha domain reaction with Cd(2+) displayed hyperbolic dependence of the observed rate constant on Cd(2+) concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd(2+) to the Zn((4-n)),Cd(n)-thiolate cluster (n = 0-3) of the alpha domain and the first order rate constant for the exchange of Cd(2+) for Zn(2+) in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn((4-n)),Cd(n)-thiolate clusters (n = 0-3) was prepared by titration of the Zn(4)-alpha domain with (113)Cd(2+). The products were analyzed by one-dimensional (113)Cd(2+) NMR spectroscopy to define the distribution of (113)Cd(2+) among the four cluster binding sites. Each of these species was also reacted with Cd(2+). The properties of these reactions were similar to those extracted from the reaction of Cd(2+) with the overall domain. Thus, the kinetic results were linked to (113)Cd(2+) occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd(2+) with the Zn(4)-alpha domain in relation to the alpha domain cluster structure.

Gold(I) efflux from auranofin-treated red blood cells: Evidence for a glutathione-gold-albumin metabolite

The efflux of gold from red blood cells (RBCs) exposed to 10-100 microM auranofin [the second generation chrysotherapy agent, triethylphosphine-(2,3,4,6-tetra-O-acetyl-1-beta-D-glucopyranosato -S-)gold(I)] was studied. RBCs in whole blood were allowed to accumulate gold, and then were placed in fresh plasma or buffered saline solution. In plasma, the kinetics of efflux were first order in gold with an apparent rate constant of 0.81 +/- 0.18 hr-1. Serum albumin, in plasma or added to a buffered solution, shifted the equilibrium between intra- and extracellular gold in favor of the latter (compared to saline solution). [14C]Glutathione, generated by in situ labeling, also effluxed and associated with the albumin and gold, providing the first direct evidence that the albumin-gold-glutathione complex (AlbSAuSG) may be a circulating metabolite of auranofin formed after both of the original ligands of auranofin are displaced.

Synthesis and characterization of complexes of trialkyl- and triarylphosphine gold(I) with thiolated purines and pyrimidines: a class of bifunctional compounds with potential antitumor activity

Abstract New complexes of the type R 3 PAuL or (R 3 PAu) 2 (μ-L) where R=ethyl or phenyl and L=6-thioguanine, 2, 6- dithioxanthine, 2, 4-dithiouracil and/or dithioerythritol have been prepared. These complexes have been identified by using elemental analysis, 1 H, 13 C and 31 P NMR spectroscopy. The structures have been proposed based on these spectroscopic studies. Sulfur appears to be the binding site in disubstituted complexes of 2, 4-dithiouracil and 1, 4-dithioerythritol, while the phosphine gold( I ) moieties appear to be S and N bonded in 2, 6-dithioxanthine and 6-thioguanine. The potential use of these complexes as antitumor drugs is discussed.

Anti-tumor activity of two binuclear gold (I) complexes with bridging dithiolate ligands

Abstract Two new binuclear complexes of gold with bridging dithiolate ligands were synthesized and characterized: (o3PAu)2(μ-DTE) and (Et3PAu)2(μ-DMTA); H2DTE=dithioerylthritol and H2DMTA=2,5-dimer- capto-1-thia-3,4-diazole. These compounds and three gold compounds with antiarthritic activity (gold sodium thiomalate, gold thioglucose and Et3PAuCl) were tested for antitumor activity using the Ehrlich- Ascites tumor cell in mice. Only (o3PAu)e(μ-DTE) showed significant activity.

Synthesis of thionato(triethylphosphine) gold(I) complexes: Analogues of “auranofin” an antiarthritic drug

New compounds having the formula [L-Au-PEt3]Cl (where L = imidazolidine-2-thione(Imt) and 1,3-Diazinane-2-thione(Diaz)) are synthesized. They are characterized by elemental analysis, infrared, 13C and 31P NMR spectroscopy.