Dual Targeting of Insulin and Venus Kinase Receptors of Schistosoma mansoni for Novel Anti-schistosome Therapy

David L Williams,Mathieu Vanderstraete,Colette Dissous,Julien Lancelot,Marion Morel,Nadège Gouignard,Katia Cailliau,Jean-François Bodart

doi:10.1371/journal.pntd.0002226

Abstract

BackgroundChemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compound has led to emergence of resistant strains of Schistosoma mansoni, therefore pointing out the necessity to find alternative drugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could represent valuable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug.Methodology/Principal FindingsSeveral commercial RTK inhibitors were tested for their potential to inhibit the kinase activities of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains (ICD) expressed in Xenopus oocytes. We measured the inhibitory effect of chemicals on meiosis resumption induced by the active ICD of the schistosome kinases in oocytes. The IR inhibitor, tyrphostin AG1024, was the most potent inhibitory compound towards SmIR and SmVKR kinases. In vitro studies then allowed us to show that AG1024 affected the viability of both schistosomula and adult worms of S. mansoni. At micromolar doses, AG1024 induced apoptosis and caused schistosomula death in a dose-dependent manner. In adult worms, AG1024 provoked alterations of reproductive organs, as observed by confocal laser scanner microscopy. With 5 µM AG1024, parasites were no more feeding and laying eggs, and they died within 48 h with 10 µM.Conclusion/SignificanceIRs and VKRs are essential in S. mansoni for key biological processes including glucose uptake, metabolism and reproduction. Our results demonstrate that inhibiting the kinase potential and function of these receptors by a single chemical compound AG1024 at low concentrations, leads to death of schistosomula and adult worms. Thus, AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosome chemotherapy.

Highlights

Schistosomiasis is the second important parasitic disease in the world
We have investigated the possibility to fight schistosomes by targeting with a single drug, insulin receptors (IRs) involved in parasite growth and metabolism and Venus Kinase Receptors (VKRs) which are unusual IRlike receptor tyrosine kinases (RTK) expressed in the parasite reproductive organs of Schistosoma mansoni
Intracellular domains (ICD) of SmIR1, SmIR2, SmVKR1 and SmVKR2 were amplified by PCR from pcDNA3.1 plasmids encoding the receptor full-length sequences [17,21,23] using Fwd 59-CCggatccAACGGAGAATTTCACGGAAACGTCTGCAG39 and Rev 59-CCctgcagTCAAATATATAAGGAAGAAGATGTGAATG-39 primers with BamH1 and Pst1 sites for SmIR1 ICD; Fwd 59-CCgaattcCGTCGTTATTATTTAAAGG TTACAGCTTGG-39 and Rev 59-CCggatccTTATGCGATAAC GTTTCTAGTTCTACTTAG-39 primers with EcoRI and BamH1 sites for SmIR2 ICD; Fwd 59-GGgaattcGTCAACCATATGAAAACCTTTG-39 and Rev 59-CCctgcagTCAAGGTAGAAACGCTAAACTGTTATC-39 primers with EcoR1 and Pst1 sites for SmVKR1 ICD; Fwd 59-AATggatccTAAACGGT CTTCCTACCGGAAAG-39 and Rev 59-CCccatggCGACGTAAACTGAAAGAAATTGAAAATCG-39 primers with BamH1 and Nco1 sites for SmVKR2 ICD

Summary

Introduction

Schistosomiasis is the second important parasitic disease in the world. Treatment relies essentially on the use of Praziquantel (PZQ), a safe and affordable drug effective against the three major human schistosome species and recommended by WHO to reduce morbidity and mortality caused by this disease. Chemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compound has led to emergence of resistant strains of Schistosoma mansoni, pointing out the necessity to find alternative drugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could represent valuable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug

Methods

Results

Discussion

Conclusion