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.

Advances in Understanding Leishmania Pathobiology: What Does RNA-Seq Tell Us?

“…Sinefungin is a naturally occurring nucleoside isolated from Streptomyces griseolus and S. incarnates bacteria with structural similarities with S-adenosyl-1-methionine ( Bachrach et al, 1980 ; Dube et al, 1983 ; McNally and Agabian, 1992 ). Sinefungin has antitrypanosomal activity and inhibits SL trans -splicing in these organisms ( Bachrach et al, 1980 ; Dube et al, 1983 ; McNally and Agabian, 1992 ; Pandarakalam et al, 2019 ).…”

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

“…Spliced leader (SL) trans -splicing is a eukaryotic post-transcriptional RNA modification whereby the 5′ end of a pre-mRNA receives a short “leader” exon from a non-coding RNA molecule that originates from elsewhere in the genome [ 1 , 2 ]. This mechanism was first discovered in trypanosomes [ 3 ] and has received much attention as a potential target for diagnosis and control of a range of medically and agriculturally important pathogens [ 1 , 4 , 5 ]. SL trans -splicing is broadly distributed among many eukaryotic groups, for example euglenozoans, dinoflagellates, cnidarians, ctenophores, platyhelminths, tunicates and nematodes, but is absent from vertebrates, insects, plants and fungi [ 2 ].…”