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Identification of annotated bioactive molecules that impair motility of the blood fluke Schistosoma mansoni.

ABSTRACT: Neglected tropical diseases are of growing worldwide concern and schistosomiasis, caused by parasitic flatworms, continues to be a major threat with more than 200 million people requiring preventive treatment. As praziquantel (PZQ) remains the treatment of choice, an urgent need for alternative treatments motivates research to identify new lead compounds that would complement PZQ by filling the therapeutic gaps associated with this treatment. Because impairing parasite neurotransmission remains a core strategy for control of parasitic helminths, we screened a library of 708 compounds with validated biological activity in humans on the blood fluke Schistosoma mansoni, measuring their effect on the motility on schistosomulae and adult worms. The primary phenotypic screen performed on schistosomulae identified 70 compounds that induced changes in viability and/or motility. Screening different concentrations and incubation times identified molecules with fast onset of activity on both life stages at low concentration (1 μM). To complement this study, similar assays were performed with chemical analogs of the cholinomimetic drug arecoline and the calcilytic molecule NPS-2143, two compounds that rapidly inhibited schistosome motility; 17 arecoline and 302 NPS-2143 analogs were tested to enlarge the pool of schistosomicidal molecules. Finally, validated hit compounds were tested on three functionally-validated neuroregulatory S. mansoni G-protein coupled receptors (GPCRs): Sm5HTR (serotonin-sensitive), SmGPR2 (histamine) and SmD2 (dopamine), revealing NPS-2143 and analogs as potent inhibitors of dopamine/epinine responses on both human and S. mansoni GPCRs. This study highlights the potential for repurposing known human therapeutic agents for potential schistosomicidal effects and expands the list of hits for further progression.

SUBMITTER: Duguet TB

PROVIDER: S-EPMC7284125 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins.We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.

Project description:RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1alpha), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced ( approximately 1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

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Identification of annotated bioactive molecules that impair motility of the blood fluke Schistosoma mansoni.

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