A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing

George Cherian Pandarakalam, Michael Speake, Stuart McElroy, Ammar Alturkistani, Lucas Philippe, Jonathan Pettitt, Berndt Müller (Lead / Corresponding author), Bernadette Connolly

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Abstract

Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity. The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs. Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs. We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.

Original languageEnglish
Pages (from-to)28-37
Number of pages10
JournalInternational Journal for Parasitology: Drugs and Drug Resistance
Volume10
Early online date5 Apr 2019
DOIs
Publication statusPublished - Aug 2019

Fingerprint

Spliced Leader RNA
Trans-Splicing
Gene Targeting
Anthelmintics
Gene Expression
sinefungin
Pharmaceutical Preparations
Parasites
Caenorhabditis elegans
Livestock
Neglected Diseases
Nematode Infections
Parasitic Diseases
Methyltransferases
Green Fluorescent Proteins
Reporter Genes
Acoustics
Fertility
Economics

Keywords

  • Anthelminitics
  • Caenorhabditis elegans
  • HTS assay
  • SL1 trans-splicing
  • Sinefungin

Cite this

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title = "A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing",
abstract = "Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity. The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs. Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs. We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.",
keywords = "Anthelminitics, Caenorhabditis elegans, HTS assay, SL1 trans-splicing, Sinefungin",
author = "Pandarakalam, {George Cherian} and Michael Speake and Stuart McElroy and Ammar Alturkistani and Lucas Philippe and Jonathan Pettitt and Berndt M{\"u}ller and Bernadette Connolly",
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A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing. / Pandarakalam, George Cherian; Speake, Michael; McElroy, Stuart; Alturkistani, Ammar; Philippe, Lucas; Pettitt, Jonathan; Müller, Berndt (Lead / Corresponding author); Connolly, Bernadette.

In: International Journal for Parasitology: Drugs and Drug Resistance, Vol. 10, 08.2019, p. 28-37.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing

AU - Pandarakalam, George Cherian

AU - Speake, Michael

AU - McElroy, Stuart

AU - Alturkistani, Ammar

AU - Philippe, Lucas

AU - Pettitt, Jonathan

AU - Müller, Berndt

AU - Connolly, Bernadette

N1 - The work was supported by the Biotechnology and Biological Sciences Research Council [Project grant BB/J007137/1] and a Medical Research Council (MRC) Confidence in Concept (2014) - University of Aberdeen Award (MC_PC_14114v.2). AA was supported by a PhD stipend from the Saudi Arabia Cultural Bureau. Some strains were provided by the CGC, which is funded by the National Institutes of Health. Office of Research Infrastructure Programs (P40 OD010440).

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N2 - Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity. The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs. Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs. We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.

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