Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target

Abstract

Background: Schistosomiasis--infection with helminth parasites in the genus Schistosoma, including S. mansoni--is a widespread, devastating tropical disease affecting more than 200 million people. No vaccine is available, and praziquantel, the only drug extensively utilized, is currently administered more than 100 million people yearly. Because praziquantel resistance may develop it is essential to identify novel drug targets. Our goal was to investigate the potential of a unique, selenium-containing parasite enzyme thioredoxin glutathione reductase (TGR) as a drug target.

Methods and findings: Using RNA interference we found that TGR is essential for parasite survival; after silencing of TGR expression, in vitro parasites died within 4 d. We also found that auranofin is an efficient inhibitor of pure TGR (Ki = 10 nM), able to kill parasites rapidly in culture at physiological concentrations (5 microM), and able to partially cure infected mice (worm burden reductions of ~60%). Furthermore, two previously used antischistosomal compounds inhibited TGR activity, suggesting that TGR is a key target during therapy with those compounds.

Conclusions: Collectively, our results indicate that parasite TGR meets all the major criteria to be a key target for antischistosomal chemotherapy. To our knowledge this is the first validation of a Schistosoma drug target using a convergence of both genetic and biochemical approaches.

Figure 1. Redox Pathways in Mammals and S. mansoni

In mammals (upper pathway), electrons from NADPH are transferred to an oxidoreductase flavoenzyme, either thioredoxin reductase (TrxR) or glutathione reductase (GR). Electrons are then transferred from the oxidoreductase flavoenzyme to the appropriate electron carrier, either oxidized thioredoxin (Trx-S₂) or glutathione disulfide (GSSG) converting them to reduced thioredoxin (Trx-[SH]₂) or glutathione (GSH), respectively. Trx-(SH)₂ and GSH then supply reducing equivalents for a number of different reactions, including those that are glutaredoxin (Grx)-dependent. In S. mansoni (lower pathway), TrxR and GR are replaced with a unique oxidoreductase flavoenzyme, TGR, which provides reducing equivalents for Trx-, GSH- and Grx-dependent reactions.

Figure 2. Naphthazarin Is a Substrate and Inhibitor of TGR

(A) Activity of recombinant S. mansoni TGR with naphthazarin as a substrate was determined by NADPH consumption under steady-state conditions. (B) Time-dependent inhibition of TrxR activity of TGR by naphthazarin with DTNB as a substrate. Concentrations of naphthazarin are 0.5 μM (♦), 1 μM (█), 2.5 μM (▴), 5 μM (×), and 10 μM (+). (C) Time-dependent inhibition of TGR activity with GSSG. Concentrations of naphthazarin are 0.5 μM (♦), 1 μM (█), 2.5 μM (▴), 5 μM (×), and 10 μM (+).

Figure 3. Inhibition of S. mansoni TGR by PAT and OPZ

Reciprocal plots showing inhibition of the TrxR and GR activities of TGR by PAT (A) and OPZ (B). Both are noncompetitive inhibitors, altering the V_max while the K _m for substrates remains constant. The apparent V_max for various concentrations of inhibitors were used to calculate K _i values. Concentrations of PAT are no drug (♦), 10 nM (), 30 nM (▴), and 75 nM (×); and of OPZ are no drug (♦), 50 μM (), 75 μM (▴), and 100 μM (×). Concentrations of DTNB ranged from 50 to 1,000 μM (TrxR activity) and of GSSG from 10 to 100 μM (GR activity).

Figure 4. Time-Dependent Inactivation of S. mansoni TGR by OPZ

The time dependency of inactivation of S. mansoni TGR (15 pmol/μl) was revealed by determining the residual GR (A) and TrxR (C) activities of enzyme in the presence of 100 μM NADPH and inhibitor at 0, 2, 4, 10, and 30 min incubation periods. OPZ concentrations were 0 (•), 25 (□), 50 (♦), 75 (Δ), and 100 (▴) μM. All incubation mixtures included 2% final DMSO. The k _obs data as a function of inhibitor concentration from the GR (B) or TrxR assay (D) versus OPZ concentrations followed a polynomial equation (not shown), suggesting that more than one binding site of OPZ is involved in the presence of different enzyme species of reacted and unreacted enzymes in mixture during the course of the inactivation process. The error bars in (B) and (D) represent ± the standard errors of the k _obs values estimated separately at each inhibitor concentration, as obtained from nonlinear regression analysis.

Figure 5. The Activity of Auranofin against Cultured Schistosoma mansoni

Each point is the average of three independent experiments ± the standard deviation. (A) Specific enzyme activities of TrxR, GR, LDH, and GPx in worm homogenates from control worms (solid lines) and worms treated with 10 μM AF (dashed lines). (B) Ratio of GSH:GSSG in worm homogenate from control worms (solid line) and worms treated with 10 μM AF (dashed line). Each assay was done in triplicate and each experiment was done three times. The error bars show the standard deviation of the three replicate experiments.

Figure 6. Survival of Schistosomula, Adult Worms, and Myeloma Cells in the Presence of Auranofin or Potassium Antimonyl Tartrate

(A) Cultured schistosomula treated with 0 μM AF (dashed line); AF 0.5 μM (♦); 1 μM (*); 2 μM (x); 5 μM (▴); 10 μM (). (B) Survival of adult Schistosoma mansoni worms at 24 h (dashed line), myeloma cells at 24 h (); and myeloma cells at 5 d (♦) at given concentrations of AF. (C) As in (B) in the presence of given concentrations of PAT. Each treatment was done in triplicate and each experiment was done three times. Error bars show the standard deviation of the three replicate experiments.

Figure 7. Survival of Schistosomula after RNAi Silencing of TGR Expression

(A) Qualitative measure of TGR transcripts by reverse transcription PCR. The abundance of TGR mRNA was greatly reduced by dsRNA treatment while the control gene GAPDH was unaffected. (B) Parasites were cultured in the presence of double-stranded TGR RNA (dashed lines) or of double-stranded irrelevant RNA (solid lines) in 20% O₂ (♦) or 0% O₂ (). A 500-nucleotide fragment (bp 1364–1866) of S. mansoni TGR cloned into PCRII-TOPO vector was used to transcribe TGR dsRNA. Irrelevant, nonschistosome dsRNA used for negative controls was synthesized from the PCRII-TOPO vector using T7 and SP6 RNA polymerases. (C) Combination of RNAi and drug treatments. Solid lines are irrelevant dsRNA treatments and dashed lines are dsTGR treatments; no additions (♦), 2 μM praziquantel () and 2 μM AF (▴). At each time point, data comparing the combination treatment (2 μM AF + TGR dsRNA) to either 2 μM AF or TGR dsRNA alone were statistically significant (*p < 0.004). In each experiment for each time point at least 1,500 parasites were scored as alive or dead (three replicates of 500 parasites). The error bars represent the standard deviations of three independent experiments.

Figure 8. Photomicrographs (100×) of Irrelevant dsRNA-Treated Schistosomula (left image) and TGR dsRNA-Treated Schistosomula (right image) after Three Days of Treatment

All organisms in the left image are alive; parasites have different shapes, elongated, contracted, and curved during movement. In the right image, all of the parasites are dead and have roughly the same shape (no movement) and internal vacuoles (arrows). The bar represents 250 μm.

Figure 9. Metabolism of Oltipraz

Metabolism of OPZ results in the production of a minor oxo analog (2), and the major dimethylated pyrrolopyrazine (3) through the biological methylation of the intermediate pyrrolopyrazine thione (4).