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 ( K _i = 10 nM), able to kill parasites rapidly in culture at physiological concentrations (5 μM), 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.

Abstract

Using both genetic and biochemical approaches, David Williams and colleagues show that the parasite thioredoxin glutathione reductase meets all the major criteria to be a key target for antischistosomal chemotherapy.

Editors' Summary

Background.

More than 200 million people are infected with schistosomes, a type of parasitic worm. Schistosomes have a complex life cycle that starts with them reproducing in freshwater snails. The snails release free-swimming, infectious parasites that burrow into the skin of people who swim in the contaminated water. Once in the human host, the parasites turn into larvae and migrate to the liver where they become juvenile worms. These mature into 10- to 20-mm-long adult worms and take up long-term residence in the veins draining the gut (Schistosoma mansoni and S. japonicum) or bladder (S. haematobium). Here, the worms mate and release eggs, some of which pass into the feces and so back into water where they hatch and infect fresh snails. Schistosomiasis causes serious health problems (including chronic liver, gut, bladder, and spleen damage) in about 20 million people, making it a disease of great public-health and socioeconomic importance in the developing countries in which it mainly occurs.

Why Was This Study Done?

The only drug available to treat schistosomiasis is praziquantel. Although it is very effective, people regularly get reinfected and need to be retreated once or twice a year. All told, 100 million people are currently being treated with praziquantel. Reliance on a single drug, however, is problematic, as the parasites are likely to develop resistance to the drug over time. The identification of new drug targets in schistosomes is therefore an urgent goal. In this study, the researchers have investigated whether thioredoxin glutathione reductase (TGR), a parasitic enzyme with several functions, might be a key target for antischistosomal chemotherapy. They chose this enzyme because adult worms need to make antioxidants (chemicals that prevent oxygen from damaging cells) to protect themselves against the human immune response. Antioxidant production in these worms depends on TGR; in mammalian cells, two specialized enzymes do its job. The researchers reasoned, therefore, that TGR might be an essential parasite protein and a potentially important drug target.

What Did the Researchers Do and Find?

The researchers made large quantities of pure TGR and tested its activity against various substrates. The enzymatic properties and substrate preferences of TGR, they found, differed somewhat from those of its mammalian counterparts. They then screened different types of compounds for their ability to inhibit TGR. Praziquantel had no effect on TGR activity, but two antischistosomal compounds that are no longer used, potassium antimonyl tartrate and oltipraz, inhibited the enzyme. The most potent inhibitor of TGR, however, was a gold-containing complex called auranofin, low levels of which inhibited TGR in test tubes, completely killed larval, juvenile, and adult parasites living in laboratory dishes within hours, and more than halved the worm burden in infected mice. Finally, the researchers used a technique called RNA silencing to test the importance of TGR for worm survival. Fragments of double-stranded RNA (dsRNA) stop proteins being made from messenger RNA that contains an identical sequence. The addition of TGR dsRNA to larval parasites in a dish greatly reduced TGR enzyme activity and killed nearly all the parasites within days.

What Do These Findings Mean?

These findings suggest TGR as a key target for antischistosomal drug development. Indeed, the discovery that two previously used antischistosomal compounds inhibit TGR suggests that the enzyme has already served as a target protein. The RNA silencing experiment shows that TGR is essential for parasite survival, and the biochemical analyses indicate that TGR and its mammalian counterparts have different substrate specificities. Thus, it should be possible to find compounds that inhibit TGR but have much less effect on the mammalian enzymes. This is certainly true for auranofin, a drug used to treat rheumatoid arthritis. Whether auranofin will be an effective treatment for schistosomiasis remains to be seen—an agent that completely kills schistosomes in animals would be preferable. However, even a 50% reduction in worm burden would decrease the human health problems caused by schistosomiasis, and a combination of auranofin (or another TGR inhibitor) with an agent that works by a different mechanism might be more effective and would also reduce the chances of the parasite developing drug resistance.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040206.

• World Health Organization provides information on schistosomiasis, including a fact sheet in English, Spanish, French, Arabic, Chinese, and Russian

• US Centers for Disease Control and Prevention provide information for the public and for professionals on schistosomiasis

• MedlinePlus encyclopedia includes an entry on schistosomiasis (in English and Spanish)

• The Schistosomiasis Control Initiative has information on the disease and its control

• Wikipedia has a page on schistosomiasis that is available in several languages (note: Wikipedia is a free online encyclopedia that anyone can edit)