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

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. Author summary: Schistosomiasis is a neglected tropical disease which affects over 200 million people and there is only one licensed drug, praziquantel, currently available for treatment. In a search for new drugs to control schistosomiasis, we tested the anti-schistosome efficacy of a series of ruthenium compounds and found that a number of them were able to inhibit parasite eggs from hatching and kill adult worms and praziquantel-refractory juvenile worms in vitro. We demonstrated that the compounds inhibit schistosome acetylcholinesterase (the enzyme that breaks down the neurotransmitter acetylcholine), which could potentially result in paralysis of the parasite, likely due to uncontrolled neuromuscular function caused by acetylcholine excess. Moreover, we showed that drug-treated worms had a significantly reduced ability to uptake exogenous glucose and markedly depleted glycogen stores, presumably through inhibition of the acetylcholinesterase-mediated glucose scavenging pathway. Lastly, we found that two of the drugs—Rubb12-tri and Rubb7-tnl—when used to treat schistosome-infected mice, were able to reduce worm burdens and significantly affect the viability of parasite eggs in vivo, which would have a marked impact on disease transmission. We believe that these complexes are desirable drug lead scaffolds which could be used to develop effective and selective compounds to control and treat schistosomiasis and, potentially, other parasitic diseases.Madhu K. Sundaraneedi, Bemnet A. Tedla, Ramon M. Eichenberger, Luke Becker, Darren Pickering, Michael J. Smout, Siji Rajan, Phurpa Wangchuk, F. Richard Keene, Alex Loukas, J. Grant Collins, Mark S. Pearso

Polypyridylruthenium(II) complexes exert in vitro and in vivo nematocidal activity and show significant inhibition of parasite acetylcholinesterases

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50 = 3.1 ± 0.4 μM) and adult (IC50 = 5.2 ± 0.3 μM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.Madhu Sundaraneedi, , Ramon M. Eichenberger, Rafid Al-Hallaf, Dai Yang, Javier Sotillo, Siji Rajan, Phurpa Wangchuk, Paul R. Giacomin, F. Richard Keene, Alex Loukas, J. Grant Collinsa, Mark S. Pearso

Synthesis and biological properties of tetranuclear ruthenium complexes containing the bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane ligand

Linear and non-linear tetranuclear ruthenium(II) complexes containing the bridging ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane have been synthesised and their biological properties examined. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the ruthenium(II) complexes were determined against six strains of bacteria: Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA); and the Gram-negative Escherichia coli (E. coli) strains MG1655, APEC, UPEC and Pseudomonas aeruginosa (P. aeruginosa). The results showed that both tetranuclear complexes had significant antimicrobial activity, with the non-linear (branched) species (Rubb7-TNL) having slightly higher activity than the corresponding linear analogue (Rubb7-TL). The corresponding toxicity against three eukaryotic cell lines − BHK (baby hamster kidney), Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma) and Hep-G2 (liver carcinoma) − have also been determined. Interestingly, both Rubb7-TNL and Rubb7-TL were as toxic to the eukaryotic cells as they were to the bacteria, a rarity for kinetically-inert cationic polypyridylruthenium(II) complexes, and exhibited lower IC50 values than cisplatin over 24-, 48- or 72-hour incubation times. Fluorescence spectroscopy was used to study the binding of the ruthenium complexes with human serum albumin (HSA). Rubb7-TNL and Rubb7-TL exhibited strong HSA binding, with equilibrium binding constants in the order of 107 M-1. Confocal microscopy was used to examine the cellular localisation of Rubb7-TNL in BHK cells. The results indicated that the ruthenium complex localised in the nucleolus. Significant accumulation was also observed in the cytoplasm, but not in the mitochondria. Taken together, the results of this study suggest that Rubb7-TNL is an unlikely candidate as an antimicrobial agent, but may have potential as an anticancer drug

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

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.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.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

Effect of Rubb₁₂-tri and Rubb₁₆-tnl on adult <i>S</i>. <i>mansoni</i> glucose uptake and storage ability.

<p>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 <i>S</i>. <i>mansoni</i> worms. (B) Levels of glycogen in extracts made from control and treated <i>S</i>. <i>mansoni</i> worms. For all assays, data are the average of triplicate biological and technical experiments ± SE. Differences were measured by ANOVA. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001, **** <i>P</i> ≤ 0.0001. (C) Scanning electron micrographs of adult male <i>S</i>. <i>mansoni</i> worm tegument after incubation with 5 μM Rubb₁₂–tri; (I) intact tubercles of control worms; (II) withered tubercules of treated worms.</p

Effect of ruthenium complexes on AChE activity in adult <i>S</i>. <i>mansoni</i> extracts.

<p>Concentration-dependent inhibition of AChE activity in <i>S</i>. <i>mansoni</i> 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 <i>S</i>. <i>mansoni</i> adult extracts in the presence of Rubb₁₂-tri. Data represent the average of triplicate experiments ± SE.</p

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.

<p>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.</p

Potency of selected ruthenium complexes against <i>S</i>. <i>mansoni</i> schistosomula after 48 h treatment.

<p>Potency of selected ruthenium complexes against <i>S</i>. <i>mansoni</i> schistosomula after 48 h treatment.</p