title: Application of RNAi to Genomic Drug Target Validation in Schistosomes.
abstract: Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these kinases may not represent suitable drug targets.
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Guidi
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Nuha R
authors.initials: NR
authors.lastname: Mansour
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Ross A
authors.initials: RA
authors.lastname: Paveley
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Carruthers
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Besnard
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Hopkins
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Ian H
authors.initials: IH
authors.lastname: Gilbert
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Quentin D
authors.initials: QD
authors.lastname: Bickle
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D055808:Drug Discovery; D004912:Erythrocytes / Q000502:physiology; D023281:Genomics; D006801:Humans; D008297:Male; D011223:Praziquantel / Q000494:pharmacology; D064546:Protein Kinase C beta / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D034622:RNA Interference; D012550:Schistosoma mansoni / Q000187:drug effects* / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics; D000097869:Polo-Like Kinase 1
chemical_list: D002147:Calmodulin; D018797:Cell Cycle Proteins; D011223:Praziquantel; D064546:Protein Kinase C beta; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D000097869:Polo-Like Kinase 1