Application of RNAi to genomic drug target validation in schistosomes

Alessandra Guidi (Lead / Corresponding author), Nuha R. Mansour, Ross A. Paveley, Ian M. Carruthers, Jérémy Besnard, Andrew L. Hopkins, Ian H. Gilbert, Quentin D. Bickle

    Research output: Contribution to journalArticle

    14 Citations (Scopus)

    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.

    Original languageEnglish
    Article numbere0003801
    Number of pages22
    JournalPLoS Neglected Tropical Diseases
    Volume9
    Issue number5
    DOIs
    Publication statusPublished - 20 May 2015

    Fingerprint

    RNA Interference
    p38 Mitogen-Activated Protein Kinases
    Phosphotransferases
    Essential Genes
    Pharmaceutical Preparations
    Erythrocytes
    Gene Knockdown Techniques
    Phenotype
    Praziquantel
    Schistosomiasis
    Drug Discovery
    Calmodulin
    Genes
    Ovum
    Mortality

    Cite this

    Guidi, A., Mansour, N. R., Paveley, R. A., Carruthers, I. M., Besnard, J., Hopkins, A. L., ... Bickle, Q. D. (2015). Application of RNAi to genomic drug target validation in schistosomes. PLoS Neglected Tropical Diseases, 9(5), [e0003801]. https://doi.org/10.1371/journal.pntd.0003801
    Guidi, Alessandra ; Mansour, Nuha R. ; Paveley, Ross A. ; Carruthers, Ian M. ; Besnard, Jérémy ; Hopkins, Andrew L. ; Gilbert, Ian H. ; Bickle, Quentin D. / Application of RNAi to genomic drug target validation in schistosomes. In: PLoS Neglected Tropical Diseases. 2015 ; Vol. 9, No. 5.
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    Application of RNAi to genomic drug target validation in schistosomes. / Guidi, Alessandra (Lead / Corresponding author); Mansour, Nuha R.; Paveley, Ross A.; Carruthers, Ian M.; Besnard, Jérémy; Hopkins, Andrew L.; Gilbert, Ian H.; Bickle, Quentin D.

    In: PLoS Neglected Tropical Diseases, Vol. 9, No. 5, e0003801, 20.05.2015.

    Research output: Contribution to journalArticle

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    AU - Guidi, Alessandra

    AU - Mansour, Nuha R.

    AU - Paveley, Ross A.

    AU - Carruthers, Ian M.

    AU - Besnard, Jérémy

    AU - Hopkins, Andrew L.

    AU - Gilbert, Ian H.

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    AB - 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.

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