Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae.

Funder: IBERS, Aberystwyth University PhD studentshipFunder: Higher Education Funding Council for Wales (HEFCW) - Global Challenges Research FundExtracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships

Antischistosomal Properties of Sclareol and Its Heck-Coupled Derivatives:Design, Synthesis, Biological Evaluation and Untargeted Metabolomics

Sclareol, a plant-derived diterpenoid widely used as a fragrance and flavoring substance, is well-known for its promising antimicrobial and anticancer properties. However, its activity on helminth parasites has not been previously reported. Here, we show that sclareol is active against larval (IC50 ≈ 13 μM), juvenile (IC50 = 5.0 μM), and adult (IC50 = 19.3 μM) stages of Schistosoma mansoni, a parasitic trematode responsible for the neglected tropical disease schistosomiasis. Microwave-assisted synthesis of Heck-coupled derivatives improved activity, with the substituents choice guided by the Matsy decision tree. The most active derivative 12 showed improved potency and selectivity on larval (IC50 ≈ 2.2 μM, selectivity index (SI) ≈ 22 in comparison to HepG2 cells), juvenile (IC50 = 1.7 μM, SI = 28.8), and adult schistosomes (IC50 = 9.4 μM, SI = 5.2). Scanning electron microscopy studies revealed that compound 12 induced blebbing of the adult worm surface at sublethal concentration (12.5 μM); moreover, the compound inhibited egg production at the lowest concentration tested (3.13 μM). The observed phenotype and data obtained by untargeted metabolomics suggested that compound 12 affects membrane lipid homeostasis by interfering with arachidonic acid metabolism. The same methodology applied to praziquantel (PZQ)-treated worms revealed sugar metabolism alterations that could be ascribed to the previously reported action of PZQ on serotonin signaling and/or effects on glycolysis. Importantly, our data suggest that compound 12 and PZQ exert different antischistosomal activities. More studies will be necessary to confirm the generated hypothesis and to progress the development of more potent antischistosomal sclareol derivatives

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds

Anti-schistosomal activities of quinoxaline-containing compounds:From hit identification to lead optimisation

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold

Flukicidal effects of abietane diterpenoid derived analogues against the food borne pathogen Fasciola hepatica.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 µM at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 µM at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogen

Structural Requirements for Dihydrobenzoxazepinone Anthelmintics:Actions against Medically Important and Model Parasites: Trichuris muris, Brugia malayi, Heligmosomoides polygyrus, and Schistosoma mansoni

Nine hundred million people are infected with the soil-transmitted helminths Ascaris lumbricoides (roundworm), hookworm, and Trichuris trichiura (whipworm). However, low single-dose cure rates of the benzimidazole drugs, the mainstay of preventative chemotherapy for whipworm, together with parasite drug resistance, mean that current approaches may not be able to eliminate morbidity from trichuriasis. We are seeking to develop new anthelmintic drugs specifically with activity against whipworm as a priority and previously identified a hit series of dihydrobenzoxazepinone (DHB) compounds that block motility of ex vivo Trichuris muris. Here, we report a systematic investigation of the structure–activity relationship of the anthelmintic activity of DHB compounds. We synthesized 47 analogues, which allowed us to define features of the molecules essential for anthelmintic action as well as broadening the chemotype by identification of dihydrobenzoquinolinones (DBQs) with anthelmintic activity. We investigated the activity of these compounds against other parasitic nematodes, identifying DHB compounds with activity against Brugia malayi and Heligmosomoides polygyrus. We also demonstrated activity of DHB compounds against the trematode Schistosoma mansoni, a parasite that causes schistosomiasis. These results demonstrate the potential of DHB and DBQ compounds for further development as broad-spectrum anthelmintics

Modified Hederagenin Derivatives Demonstrate Ex Vivo Anthelmintic Activity against Fasciola hepatica

Infection with Fasciola hepatica (liver fluke) causes fasciolosis (or fascioliasis) and poses a considerable economic as well as welfare burden to both the agricultural and animal health sectors. Here, we explore the ex vivo anthelmintic potential of synthetic derivatives of hederagenin, isolated in bulk from Hedera helix. Thirty-six compounds were initially screened against F. hepatica newly excysted juveniles (NEJs) of the Italian strain. Eleven of these compounds were active against NEJs and were selected for further study, using adult F. hepatica derived from a local abattoir (provenance unknown). From these eleven compounds, six demonstrated activity and were further assessed against immature liver flukes of the Italian strain. Subsequently, the most active compounds (n = 5) were further evaluated in ex vivo dose response experiments against adult Italian strain liver flukes. Overall, MC042 was identified as the most active molecule and the EC50 obtained from immature and adult liver fluke assays (at 24 h post co-culture) are estimated as 1.07 μM and 13.02 μM, respectively. When compared to the in vitro cytotoxicity of MDBK bovine cell line, MC042 demonstrated the highest anthelmintic selectivity (44.37 for immature and 3.64 for adult flukes). These data indicate that modified hederagenins display properties suitable for further investigations as candidate flukicides