Polypyridylruthenium(II) complexes exert anti-schistosome activity and inhibit parasite acetylcholinesterases

Abstract

Background: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)-the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs.

Methodology/principal findings: We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri-68%, Rubb7-tnl-56%) and were significantly morphologically altered (Rubb12-tri-62% abnormal, Rubb7-tnl-35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway.

Conclusions/significance: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.

Fig 1. The kinetically inert tri-nuclear (Rubb_n-tri), linear tetra-nuclear Rubb_n-tl and non-linear tetra-nuclear Rubb_n-tnl) ruthenium(II) complexes.

Fig 2. Effect of ruthenium complexes on AChE activity in adult S. mansoni extracts.

Concentration-dependent inhibition of AChE activity in S. mansoni adult extracts when treated with Rubb₁₂-tri, a representative member of the ruthenium complexes tested, as determined by Ellman assay. (A) Dose-response curve of Rubb₁₂-tri. (B) Lineweaver-Burk inhibition plot of AChE activity in S. mansoni adult extracts in the presence of Rubb₁₂-tri. Data represent the average of triplicate experiments ± SE.

Fig 3. Activity of ruthenium complexes against adult S. mansoni worms.

(A) Selective representation of survival of parasites (cultured in Basch media) after treatment with Ru complexes (50 μM). Data represents the average of duplicate experiments ± SE. (B) Survival of parasites (cultured in Basch media) after treatment with various concentrations of the most potent ruthenium complexes determined from the screening experiment. Data represents the average of duplicate experiments ± SE. (C) Alteration in general morphology of adult S. mansoni worms caused by ruthenium complexes. I: control parasites; II: parasites treated with Rubb₁₂-tri.

Fig 4. Inhibition of S. mansoni egg hatching and effect on egg development by ruthenium complexes.

(A) Graph representing the percentage of S. mansoni eggs hatched (motility index) in the presence of various ruthenium complexes (50 μM) as determined by the x-WORM motility assay. Data represents the average of triplicate experiments ± SE. (B) Triplicate sets of five pairs of adult S. mansoni worms were cultured in Basch media with or without 5 μM Rubb₁₂-tri for 72 h. The eggs released into the media were counted and those that were misshapen or immature were scored as “abnormally developed”. Graph shows the difference in percentage of normally developed eggs between treated and control groups and data represents the average of triplicate experiments ± SE. Differences in egg hatching were measured by ANOVA and differences in egg development by t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001,****P ≤ 0.0001.

Fig 5. Action of Rubb₁₂-tri and Rubb₁₆-tnl on adult S. mansoni AChE and AP activity.

Five pairs of worms were cultured in Basch media for 24 h in the presence of a sub-lethal dose (5 μM) of Rubb₁₂-tri or Rubb₁₆-tnl and then incubated in AChE assay buffer or AP assay buffer. PBS extracts were then made from equal amounts of control and treated worms and 30 μg of each extract was used to determine somatic AChE activity by the Ellman method. (A) surface AChE (B) somatic AChE and (C) surface AP activity of control worms and worms treated with Rubb₁₂-tri or Rubb₁₆-tnl. For all assays, data are the average of triplicate biological and technical experiments ± SE. Differences were measured by ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001.

Fig 6. Effect of Rubb₁₂-tri and Rubb₁₆-tnl on adult S. mansoni glucose uptake and storage ability.

Worms were cultured in Basch media for 24 h in the presence of a sub-lethal dose (5 μM) of Rubb₁₂-tri or Rubb₁₆-tnl. Worms were then incubated for 24 h in DMEM containing 1 mg/ml glucose. PBS extracts were then made from equal amounts of control and treated worms and 30 ug of each extract was used to determine the glycogen content of the worms using a modified glucose oxidase assay. (A) Amount of glucose in media collected from control and treated S. mansoni worms. (B) Levels of glycogen in extracts made from control and treated S. mansoni worms. For all assays, data are the average of triplicate biological and technical experiments ± SE. Differences were measured by ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001. (C) Scanning electron micrographs of adult male S. mansoni worm tegument after incubation with 5 μM Rubb₁₂–tri; (I) intact tubercles of control worms; (II) withered tubercules of treated worms.

Fig 7. Cytotoxicity of ruthenium complexes.

Toxicity against human bile duct cells (H69) after 72 h incubation with Rubb₁₂-tri or Rubb₇-tnl, praziquantel and dichlorvos—an organophosphorus AChE inhibitor—as determined by the MTT cell viability assay. Data are the average of six replicate experiments ± SE.

Fig 8. In vivo effect of Rubb₁₂-tri and Rubb₇-tnl on S. mansoni-infected mice.

(A) Effect of Rubb₁₂-tri and Rubb₇-tnl on adult worm burden. Symbols represent data from individual mice and are the combination of two independent trials (trial 1 PBS control—n = 8 mice, trial 1 Rubb₁₂-tri-treated—n = 8 mice, trial 1 Rubb₇-tnl-treated—n = 6 mice, trial 2 PBS control—n = 7 mice, trial 2 Rubb₁₂-tri-treated—n = 7 mice, trial 2 Rubb₇-tnl-treated—n = 8 mice). (B) Surface AChE activity of worms recovered from control and treated mice. Data are the average of triplicate technical assays ± SE on extracts made from worms (five pairs) pooled from each group of each of the two trials. (C) Hatching viability of eggs obtained from the pooled livers of control and treated mice from trial 1 (PBS control—n = 8, Rubb₁₂-tri-treated—n = 8, Rubb₇-tnl-treated—n = 6). Data are the average of ten replicate counts ± SE of hatched miracidia. (D) Eggs were harvested from triplicate sets of worms (five pairs) from a pool of each group of trial 2 (PBS control—n = 7 mice, Rubb₁₂-tri-treated—n = 7 mice, Rubb₇-tnl-treated—n = 8 mice) after culturing the parasites for 24 h in Basch media and the percentage of mature, morphologically “normal” eggs released from worms recovered from control and treated mice was assessed. Data are the average counts ± SE of eggs released from triplicate sets (five pairs) of worms from trial 2. Differences were measured by ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001. (E) Auto-fluorescence images (20×) of eggs released from worms recovered from (I and II) control and (III and IV) treated mice.