Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development.

Schistosomiasis is a disease affecting >200 million people worldwide, but its treatment relies on a single agent, praziquantel. To investigate new avenues for schistosomiasis control, we have conducted the first systematic analysis of bromodomain-containing proteins (BCPs) in a causative species, Schistosoma mansoni. Having identified 29 putative bromodomains (BRDs) in 22 S. mansoni proteins, we selected SmBRD3, a tandem BRD-containing BCP that shows high similarity to the human bromodomain and extra terminal domain (BET) family, for further studies. Screening 697 small molecules identified the human BET BRD inhibitor I-BET726 as a ligand for SmBRD3. An X-ray crystal structure of I-BET726 bound to the second BRD of SmBRD3 [SmBRD3(2)] enabled rational design of a quinoline-based ligand (15) with an ITC Kd = 364 ± 26.3 nM for SmBRD3(2). The ethyl ester pro-drug of compound 15 (compound 22) shows substantial effects on sexually immature larval schistosomula, sexually mature adult worms, and snail-infective miracidia in ex vivo assays.

Chemical modulation of Schistosoma mansoni lysine specific demethylase 1 (SmLSD1) induces wide-scale biological and epigenomic changes.

Background: Schistosoma mansoni, a parasitic worm species responsible for the neglected tropical disease schistosomiasis, undergoes strict developmental regulation of gene expression that is carefully controlled by both genetic and epigenetic processes. As inhibition of S. mansoni epigenetic machinery components impairs key transitions throughout the parasite's digenetic lifecycle, a greater understanding of how epi-drugs affect molecular processes in schistosomes could lead to the development of new anthelmintics. Methods:   In vitro whole organism assays were used to assess the anti-schistosomal activity of 39 Homo sapiens Lysine Specific Demethylase 1 (HsLSD1) inhibitors on different parasite life cycle stages. Moreover, tissue-specific stains and genomic analysis shed light on the effect of these small molecules on the parasite biology. Results: Amongst this collection of small molecules, compound 33 was the most potent in reducing ex vivo viabilities of schistosomula, juveniles, miracidia and adults. At its sub-lethal concentration to adults (3.13 µM), compound 33 also significantly impacted oviposition, ovarian as well as vitellarian architecture and gonadal/neoblast stem cell proliferation. ATAC-seq analysis of adults demonstrated that compound 33 significantly affected chromatin structure (intragenic regions > intergenic regions), especially in genes differentially expressed in cell populations (e.g., germinal stem cells, hes2 + stem cell progeny, S1 cells and late female germinal cells) associated with these ex vivo phenotypes. KEGG analyses further highlighted that chromatin structure of genes associated with sugar metabolism as well as TGF-beta and Wnt signalling were also significantly perturbed by compound 33 treatment. Conclusions: This work confirms the importance of histone methylation in S. mansoni lifecycle transitions, suggesting that evaluation of LSD1 - targeting epi-drugs may facilitate the search for next-generation anti-schistosomal drugs. The ability of compound 33 to modulate chromatin structure as well as inhibit parasite survival, oviposition and stem cell proliferation warrants further investigations of this compound and its epigenetic target SmLSD1.

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.

Using ChEMBL to Complement Schistosome Drug Discovery.

Schistosomiasis is one of the most important neglected tropical diseases. Until an effective vaccine is registered for use, the cornerstone of schistosomiasis control remains chemotherapy with praziquantel. The sustainability of this strategy is at substantial risk due to the possibility of praziquantel insensitive/resistant schistosomes developing. Considerable time and effort could be saved in the schistosome drug discovery pipeline if available functional genomics, bioinformatics, cheminformatics and phenotypic resources are systematically leveraged. Our approach, described here, outlines how schistosome-specific resources/methodologies, coupled to the open-access drug discovery database ChEMBL, can be cooperatively used to accelerate early-stage, schistosome drug discovery efforts. Our process identified seven compounds (fimepinostat, trichostatin A, NVP-BEP800, luminespib, epoxomicin, CGP60474 and staurosporine) with ex vivo anti-schistosomula potencies in the sub-micromolar range. Three of those compounds (epoxomicin, CGP60474 and staurosporine) also demonstrated potent and fast-acting ex vivo effects on adult schistosomes and completely inhibited egg production. ChEMBL toxicity data were also leveraged to provide further support for progressing CGP60474 (as well as luminespib and TAE684) as a novel anti-schistosomal compound. As very few compounds are currently at the advanced stages of the anti-schistosomal pipeline, our approaches highlight a strategy by which new chemical matter can be identified and quickly progressed through preclinical development.

Perspective on Schistosomiasis Drug Discovery: Highlights from a Schistosomiasis Drug Discovery Workshop at Wellcome Collection, London, September 2022.

In September 2022, the Drug Discovery Unit at the University of Dundee, UK, organised an international meeting at the Wellcome Collection in London to explore the current clinical situation and challenges associated with treating schistosomiasis. The aim of this meeting was to discuss the need for new treatments in view of the clinical situation and to ascertain what the key requirements would be for any potential new anti-schistosomals. This information will be essential to inform ongoing drug discovery efforts for schistosomiasis. We also discussed the potential drug discovery pathway and associated criteria for progressing compounds to the clinic. To date, praziquantel (PZQ) is the only drug available to treat all species causing schistosomiasis, but it is often unable to completely clear parasites from an infected patient, partially due to its inactivity against juvenile worms. PZQ-mediated mass drug administration campaigns conducted in endemic areas (e.g., sub-Saharan Africa, where schistosomiasis is primarily prevalent) have contributed to reducing the burden of disease but will not eliminate the disease as a public health problem. The potential for Schistosoma to develop resistance towards PZQ, as the sole treatment available, could become a concern. Consequently, new anthelmintic medications are urgently needed, and this Perspective aims to capture some of the learnings from our discussions on the key criteria for new treatments.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

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 pathogens.

Identification of anti-schistosomal, anthelmintic and anti-parasitic compounds curated and text-mined from the scientific literature.

More than a billion people are infected with parasitic worms, including nematodes, such as hookworms, and flatworms, such as blood flukes. Few drugs are available to treat worm infections, but high-throughput screening approaches hold promise to identify novel drug candidates. One problem for researchers who find an interesting 'hit' from a high-throughput screen is to identify whether that compound, or a similar compound has previously been published as having anthelmintic or anti-parasitic activity. Here, we present (i) data sets of 2,828 anthelmintic compounds, and 1,269 specific anti-schistosomal compounds, manually curated from scientific papers and books, and (ii) a data set of 24,335 potential anthelmintic and anti-parasitic compounds identified by text-mining PubMed abstracts. We provide their structures in simplified molecular-input line-entry system (SMILES) format so that researchers can easily compare 'hits' from their screens to these anthelmintic compounds and anti-parasitic compounds and find previous literature on them to support/halt their progression in drug discovery pipelines.

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<0.05; α-NAGAL > 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.

Daily rhythms in gene expression of the human parasite Schistosoma mansoni.

BACKGROUND: The consequences of the earth's daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis. RESULTS: Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host's active phase and a 'peak in metabolic activity' during the host's resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity. CONCLUSIONS: There are daily rhythms in the transcriptomes of adult S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.

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

Extracellular 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.

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. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 µM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 µM, 0.20 µM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.

Plasmacytoid Dendritic Cells Facilitate Th Cell Cytokine Responses throughout Schistosoma mansoni Infection.

Plasmacytoid dendritic cells (pDCs) are potent producers of type I IFN (IFN-I) during viral infection and respond to IFN-I in a positive feedback loop that promotes their function. IFN-I shapes dendritic cell responses during helminth infection, impacting their ability to support Th2 responses. However, the role of pDCs in type 2 inflammation is unclear. Previous studies have shown that pDCs are dispensable for hepatic or splenic Th2 responses during the early stages of murine infection with the trematode Schistosoma mansoni at the onset of parasite egg laying. However, during S. mansoni infection, an ongoing Th2 response against mature parasite eggs is required to protect the liver and intestine from acute damage and how pDCs participate in immune responses to eggs and adult worms in various tissues beyond acute infection remains unclear. We now show that pDCs are required for optimal Th2 cytokine production in response to S. mansoni eggs in the intestinal-draining mesenteric lymph nodes throughout infection and for egg-specific IFN-γ at later time points of infection. Further, pDC depletion at chronic stages of infection led to increased hepatic and splenic pathology as well as abrogated Th2 cell cytokine production and activation in the liver. In vitro, mesenteric lymph node pDCs supported Th2 cell responses from infection-experienced CD4+ T cells, a process dependent on pDC IFN-I responsiveness, yet independent of Ag. Together, these data highlight a previously unappreciated role for pDCs and IFN-I in maintaining and reinforcing type 2 immunity in the lymph nodes and inflamed tissue during helminth infection.

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.

Identifying and validating the presence of Guanine-Quadruplexes (G4) within the blood fluke parasite Schistosoma mansoni.

Schistosomiasis is a neglected tropical disease that currently affects over 250 million individuals worldwide. In the absence of an immunoprophylactic vaccine and the recognition that mono-chemotherapeutic control of schistosomiasis by praziquantel has limitations, new strategies for managing disease burden are urgently needed. A better understanding of schistosome biology could identify previously undocumented areas suitable for the development of novel interventions. Here, for the first time, we detail the presence of G-quadruplexes (G4) and putative quadruplex forming sequences (PQS) within the Schistosoma mansoni genome. We find that G4 are present in both intragenic and intergenic regions of the seven autosomes as well as the sex-defining allosome pair. Amongst intragenic regions, G4 are particularly enriched in 3´ UTR regions. Gene Ontology (GO) term analysis evidenced significant G4 enrichment in the wnt signalling pathway (p<0.05) and PQS oligonucleotides synthetically derived from wnt-related genes resolve into parallel and anti-parallel G4 motifs as elucidated by circular dichroism (CD) spectroscopy. Finally, utilising a single chain anti-G4 antibody called BG4, we confirm the in situ presence of G4 within both adult female and male worm nuclei. These results collectively suggest that G4-targeted compounds could be tested as novel anthelmintic agents and highlights the possibility that G4-stabilizing molecules could be progressed as candidates for the treatment of schistosomiasis.

Schistosoma mansoni venom allergen-like protein 6 (SmVAL6) maintains tegumental barrier function.

The Schistosoma mansoni venom allergen-like protein (SmVAL) superfamily is a collection of at least 29 molecules that have been classified into two distinctive groups (Group 1 and Group 2 SmVALs). The fundamental basis for SmVAL segregation relates to signal peptide and conserved cysteine retention (present in all Group 1 SmVALs, but absent in all Group 2 SmVALs). These structural differences have led to the hypothesis that most Group 1 SmVALs, found as components of schistosome excretory/secretory (E/S) products, predominantly interact with their environment (intermediate or definitive hosts) whereas the Group 2 SmVALs are retained within the schistosome to fulfil parasite-related functions. While experimental evidence to support Group 1 SmVAL/host interactions is growing, similar support for identification of parasite-related Group 2 SmVAL functions is currently lacking. By applying a combination of approaches to the study of SmVAL6, we provide the first known evidence for an essential function of a Group 2 SmVAL in schistosome biology. After whole mount in situ hybridisation (WISH) localised Smval6 to the anterior region of the oesophageal gland (AOG) and cells scattered through the mesenchyme in adult schistosomes, short interfering RNA (siRNA)-mediated silencing of Smval6 was employed to assess loss of function phenotypes. Here, siSmval6-mediated knockdown of transcript and protein levels led to an increase in tegumental permeability as assessed by the quantification of TAMRA-labelled dextran throughout sub-tegumental cells/tissues. Yeast two hybrid screening using SmVAL6 as a bait revealed Sm14 (a fatty acid binding protein) and a dynein light chain (DLC) as directly interacting partners. Interrogation of single-cell RNA-seq (scRNA-seq) data supported these protein interactions by demonstrating the spatial co-expression of Smval6/dlc/Sm14 in a small proportion of adult cell types (e.g. neurons, tegumental cells and neoblasts). In silico modelling of SmVAL6 with Sm14 and DLC provided evidence that opposing faces of SmVAL6 were likely responsible for these protein/protein interactions. Our results suggest that SmVAL6 participates in oesophageal biology, formation of higher order protein complexes and maintenance of tegumental barrier function. Further studies of other Group 2 SmVALs may reveal additional functions of this enigmatic superfamily.

Identification of 6-(piperazin-1-yl)-1,3,5-triazine as a chemical scaffold with broad anti-schistosomal activities.

Background: Schistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease of considerable importance in resource-poor communities throughout the developing world. In the absence of an immunoprophylactic vaccine and due to over-reliance on a single chemotherapy (praziquantel), schistosomiasis control is at risk should drug insensitive schistosomes develop. In this context, application of in silico virtual screening on validated schistosome targets has proven successful in the identification of novel small molecules with anti-schistosomal activity.   Methods: Focusing on the Schistosoma mansoni histone methylation machinery, we herein have used RNA interference (RNAi), ELISA-mediated detection of H3K4 methylation, homology modelling and in silico virtual screening to identify a small collection of small molecules for anti-schistosomal testing. A combination of low to high-throughput whole organism assays were subsequently used to assess these compounds' activities on miracidia to sporocyst transformation, schistosomula phenotype/motility metrics and adult worm motility/oviposition readouts. Results: RNAi-mediated knockdown of smp_138030/smmll-1 (encoding a histone methyltransferase, HMT) in adult worms (~60%) reduced parasite motility and egg production. Moreover, in silico docking of compounds into Smp_138030/SmMLL-1's homology model highlighted competitive substrate pocket inhibitors, some of which demonstrated significant activity on miracidia, schistosomula and adult worm lifecycle stages together with variable effects on HepG2 cells. Particularly, the effect of compounds containing a 6-(piperazin-1-yl)-1,3,5-triazine core on adult schistosomes recapitulated the results of the smp_138030/smmll-1 RNAi screens. Conclusions: The biological data and the structure-activity relationship presented in this study define the 6-(piperazin-1-yl)-1,3,5-triazine core as a promising starting point in ongoing efforts to develop new urgently needed schistosomicides.

Quorum sensing N-Acyl homoserine lactones are a new class of anti-schistosomal.

BACKGROUND: Schistosomiasis is a prevalent neglected tropical disease that affects approximately 300 million people worldwide. Its treatment is through a single class chemotherapy, praziquantel. Concerns surrounding the emergence of praziquantel insensitivity have led to a need for developing novel anthelmintics. METHODOLOGY/PRINCIPLE FINDINGS: Through evaluating and screening fourteen compounds (initially developed for anti-cancer and anti-viral projects) against Schistosoma mansoni, one of three species responsible for most cases of human schistosomiasis, a racemic N-acyl homoserine (1) demonstrated good efficacy against all intra mammalian lifecycle stages including schistosomula (EC50 = 4.7 µM), juvenile worms (EC50 = 4.3 µM) and adult worms (EC50 = 8.3 µM). To begin exploring structural activity relationships, a further 8 analogues of this compound were generated, including individual (R)- and (S)- enantiomers. Upon anti-schistosomal screening of these analogues, the (R)- enantiomer retained activity, whereas the (S)- lost activity. Furthermore, modification of the lactone ring to a thiolactone ring (3) improved potency against schistosomula (EC50 = 2.1 µM), juvenile worms (EC50 = 0.5 µM) and adult worms (EC50 = 4.8 µM). As the effective racemic parent compound is structurally similar to quorum sensing signaling peptides used by bacteria, further evaluation of its effect (along with its stereoisomers and the thiolactone analogues) against Gram+ (Staphylococcus aureus) and Gram- (Escherichia coli) species was conducted. While some activity was observed against both Gram+ and Gram- bacteria species for the racemic compound 1 (MIC 125 mg/L), the (R) stereoisomer had better activity (125 mg/L) than the (S) (>125mg/L). However, the greatest antimicrobial activity (MIC 31.25 mg/L against S. aureus) was observed for the thiolactone containing analogue (3). CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first demonstration that N-Acyl homoserines exhibit anthelmintic activities. Furthermore, their additional action on Gram+ bacteria opens a new avenue for exploring these molecules more broadly as part of future anti-infective initiatives.

Large-scale RNAi screening uncovers therapeutic targets in the parasite Schistosoma mansoni.

Schistosome parasites kill 250,000 people every year. Treatment of schistosomiasis relies on the drug praziquantel. Unfortunately, a scarcity of molecular tools has hindered the discovery of new drug targets. Here, we describe a large-scale RNA interference (RNAi) screen in adult Schistosoma mansoni that examined the function of 2216 genes. We identified 261 genes with phenotypes affecting neuromuscular function, tissue integrity, stem cell maintenance, and parasite survival. Leveraging these data, we prioritized compounds with activity against the parasites and uncovered a pair of protein kinases (TAO and STK25) that cooperate to maintain muscle-specific messenger RNA transcription. Loss of either of these kinases results in paralysis and worm death in a mammalian host. These studies may help expedite therapeutic development and invigorate studies of these neglected parasites.