Identification of oxadiazoles as new drug leads for the control of schistosomiasis

Abstract

Treatment for schistosomiasis, which is responsible for more than 280,000 deaths annually, depends almost exclusively on praziquantel. Millions of people are treated annually with praziquantel, and drug-resistant parasites thus are likely to evolve. Phosphinic amides and oxadiazole 2-oxides, identified from a quantitative high-throughput screen, were shown to inhibit a parasite enzyme, thioredoxin glutathione reductase (TGR), with activities in the low micromolar to low nanomolar range. Incubation of parasites with these compounds led to rapid inhibition of TGR activity and parasite death. The activity of the oxadiazole 2-oxides was associated with a donation of nitric oxide. Treatment of schistosome-infected mice with 4-phenyl-1,2,5-oxadiazole-3-carbonitrile-2-oxide led to marked reductions in worm burdens from treatments against multiple parasite stages and egg-associated pathologies. The compound was active against the three major schistosome species infecting humans. These protective effects exceed benchmark activity criteria set by the World Health Organization for lead compound development for schistosomiasis.

Fig. 1

Activity of phosphinic amides and oxadiazole 2-oxides against cultured Schistosoma mansoni worms. (a) Survival of cultured adult S. mansoni worms treated with phosphinic amides. Cultured worms treated with 50 μM 1 (◆), 50 μM 2 (■), 50 μM 3 (□), 25 μM 3 (□, dotted line) and 50 μM 4 (▲). (b) Survival of cultured adult S. mansoni worms treated with oxadiazole 2-oxides. Cultured worms were treated with 50 μM 5 (◆), 6 (■), 7 (▲), 8 (◇), or 10 (△), or 10 μM 9 (□). (c) NO release by oxadiazole 2-oxides (10 μM) after incubation with 15 nM S. mansoni TGR (black bars), with 15 nM S. mansoni TGR plus 100 μM NADPH (gray bars), or with 5 mM cysteine alone (open bars) was determined using ABTS method. (d) Survival of adult worms in 10 μM 9 (◇), 10 μM 9 plus 100 μM carboxy-PTIO (△), and drug carrier plus 100 μM carboxy-PTIO treated control (□). (e) Cytotoxicity after five days culture at the indicated concentrations of compounds 3 (■) and 9 (◆) and praziquantel (▲) against mouse myeloma line SP2/0 (solid lines) and adult Schistosoma mansoni worms (dashed lines). Error bars represent the ± standard deviation form three independent experiments.

Fig. 2

The action of compounds 3 and 9 on TGR activities in cultured Schistosoma mansoni worms. (a) The specific thioredoxin reductase activity of TGR in worm homogenates from control worms (◇), 50 μM 3 treated worms (□), and 10 μM 9 treated worms (△). (b) The specific glutathione reductase activity of TGR in worm homogenate from control worms (◇), 50 μM 3 treated worms (□), and 10 μM 9 treated worms (△). (c) The specific activity of lactate dehydrogenase in worm homogenates from control worms (◇), 50 μM 3 treated worms (□), and 10 μM 9 treated worms (△).The error bars represent ± standard deviation from the average of three independent experiments.

Fig. 3

In vivo drug treatment with compound 9. (a) Administration of compound 9 at 10 mg/kg for 5 days at different points during the development of Schistosoma mansoni in the mouse. (b) The effect on adult worm burdens after treatment of S. mansoni infected mice with compound 9 in treatment 1 (▲, skin-stage parasites, n= 8), treatment 2 (■, juvenile liver-stage parasites, n = 8), treatment 3 (□, Adult egg-laying parasites, n= 8); drug carrier treated control infected mice (◆, n = 16). Points represent data from individual mice; * P < 0.0001. The horizontal bars represent the average point of each treatment. (c) Pictures of the livers collected from the different treatments, the white dots are liver granulomas resulting from host immunological reaction against trapped parasite eggs. (d) Worms recovered from treatment 2 and control infected mice. Worms recovered from treatment 2 are small and stunted, especially males, relative to worms recovered from untreated mice. (e) Anti-pathology effects of treatment with compound 9. Liver (closed symbols) and spleen (open symbols) weights from infected control mice (I, ◆), treatment 1 (1, ■), treatment 2 (2, ▲) and uninfected age matched control mice (U, •); * P < 0.0001.

Fig. 3

In vivo drug treatment with compound 9. (a) Administration of compound 9 at 10 mg/kg for 5 days at different points during the development of Schistosoma mansoni in the mouse. (b) The effect on adult worm burdens after treatment of S. mansoni infected mice with compound 9 in treatment 1 (▲, skin-stage parasites, n= 8), treatment 2 (■, juvenile liver-stage parasites, n = 8), treatment 3 (□, Adult egg-laying parasites, n= 8); drug carrier treated control infected mice (◆, n = 16). Points represent data from individual mice; * P < 0.0001. The horizontal bars represent the average point of each treatment. (c) Pictures of the livers collected from the different treatments, the white dots are liver granulomas resulting from host immunological reaction against trapped parasite eggs. (d) Worms recovered from treatment 2 and control infected mice. Worms recovered from treatment 2 are small and stunted, especially males, relative to worms recovered from untreated mice. (e) Anti-pathology effects of treatment with compound 9. Liver (closed symbols) and spleen (open symbols) weights from infected control mice (I, ◆), treatment 1 (1, ■), treatment 2 (2, ▲) and uninfected age matched control mice (U, •); * P < 0.0001.