Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects

Abstract

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.

SCHEME 1.

Possible reaction path for AF and SmTGR. One molecule of AF reacts with wild type reduced SmTGR (species 1) to yield a transient intermediate bearing an Au(I) coordinated with Sec⁵⁹⁷ and triethylphosphine (species 2); we hypothesize that acetoxythioglucose is released first (see text). The intermediate species releases triethylphosphine to form a Sec-gold-Cys complex with any of the available cysteinyl residues; three reaction products are possible (species 3, 4, and 6). The chemical species in which the C-terminal arm is cross-linked via the gold ion to a distant Cys residue (species 4 and 6) may rearrange to yield the more stable short distance Cys-gold-Cys complexes (species 5 and 7). When equilibrium is reached, a mixture of species 3, 5, and 7 is expected to be present, dynamically interchanging via the less stable intermediates 4 and 6. Additional molecules of AF may be introduced, leading to SmTGR molecules bearing two or three gold ions. The gold-binding site 3, in which the metal ion is not coordinated by Cys or Sec, is not considered in the scheme.

FIGURE 1.

The three gold-binding sites of wild type SmTGR. A, the three-dimensional model of one subunit is shown with the three gold-binding sites. Site 1 shows the gold in between Cys¹⁵⁴ and Cys¹⁵⁹; site 2 shows the gold in between Cys⁵²⁰ and Cys⁵⁷⁴; site 3 shows the gold in the putative NADPH-binding pocket. The glutaredoxin domain of TGR is shown in green, whereas the thioredoxin domain is shown in red. The bound flavin is also highlighted. B, site 1. The linear geometry of the Cys¹⁵⁴-gold-Cys¹⁵⁹ adduct is shown. The occupancy of the gold atom is about 50%. Both distances of the sulfur-gold bond are 2.3 Å, as expected for this type of coordination moiety. C, site 2. The electron density map (2F_o − F_c) contoured at 1 σ shows the possible charge transfer complex between the gold and Phe⁵⁰⁵. D, site 2. The gold atom between Cys⁵⁷⁴ and Cys⁵²⁰ is shown together with the other residues that surround the metal, i.e. Phe⁵⁰⁵, Pro⁵⁰⁷, and Pro⁵⁴². E, site 3. Gold in the putative NADPH-binding site of SmTGR. Tyr²⁹⁶ is known to swing upon NADPH binding in thiol reductase enzymes (30). Ser²⁹⁵ is the residue closest to the gold (Ser²⁹⁵(OG)-gold: 3.2 Å). Other van der Waals' contacts are with the main chain atoms of the polypeptide (Ala³⁹⁰, Val³⁹¹, Gly³⁹², and Arg³⁹³).

FIGURE 2.

The SmTGR crystal structure in complex with gold ions (in green) is superimposed to the mouse TR with NADPH bound (in magenta; Protein Data Bank code 1zdl (30)). The root mean square deviation is 0.82 Å over the 462 aligned residues. The residues surrounding NADPH in mouse TR are conserved in SmTGR (sequence alignment not shown). The structural comparison shows the change in conformation of the loop 293–296 and in particular of Tyr²⁹⁶ and Ser²⁹⁵, highlighted for the two enzymes as balls and sticks (the other amino acid side chains are omitted for clarity). The OG atom of Ser²⁹⁵ is the closest contact with the gold ion in the SmTGR crystal structure (see “Results” and Fig. 1). In the mouse TR structure, Ser²⁹⁵ shifts in position to make room for the bound NADPH; the clash between the metal in site 3 and the phosphate of the cofactor in SmTGR is self-evident.

FIGURE 3.

A, baker's yeast GR is inactivated by AF: time courses obtained at concentrations of 1 μm (squares), 4 μm (closed circles), 10 μm (open circles), and 50 μm (open triangles). B, effect of BzSe on baker's yeast GR inactivation by AF. Time course in the presence of 4 μm AF (circles) or 4 μm AF plus 2 μm BzSe (triangles). GR exposed to a mixture in which AF 4 μm and BzSe 2 μm were preincubated for 2 h before the assay (pentagons). C, inactivation by AF of truncated SmTGR and wild type SmTGR, and the effect of BzSe. Time courses of truncated SmTGR in the presence of 8 μm AF (circles) or 8 μm AF plus 3 μm BzSe (triangles). Truncated SmTGR exposed to a mixture in which AF 8 μm and BzSe 3 μm were preincubated for 2 h before the assay (pentagons); time course of wild type SmTGR 20 nm incubated with 50 nm AF (open circles).

FIGURE 4.

Rate constant of inactivation (min⁻¹) of yeast GR (0.5 μm) as a function of AF concentration (1, 4, 10, and 50 μm), as derived from linearization of the first points of the time courses reported for Fig. 3A.