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

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).

PY - 2019/8

Y1 - 2019/8

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.

AB - 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.

KW - Anthelminitics

KW - Caenorhabditis elegans

KW - HTS assay

KW - SL1 trans-splicing

KW - Sinefungin

UR - https://www.scopus.com/pages/publications/85064465483

U2 - 10.1016/j.ijpddr.2019.04.001

DO - 10.1016/j.ijpddr.2019.04.001

M3 - Article

C2 - 31015150

SN - 2211-3207

VL - 10

SP - 28

EP - 37

JO - International Journal for Parasitology: Drugs and Drug Resistance

JF - International Journal for Parasitology: Drugs and Drug Resistance

ER -