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.

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.

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.

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.

The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets.

BACKGROUND: Praziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium. METHODOLOGY/PRINCIPLE FINDINGS: Thirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 µM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor). CONCLUSION/SIGNIFICANCE: Collectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.

Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11.

Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, with a prevalence of up to 12% of women of reproductive age, and is in part characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium. A high percentage of PCOS patients suffer from infertility, a condition that appears to be linked to mistimed and incomplete decidualisation critically affecting events surrounding embryo implantation. The aim of this study was to examine the involvement of MAGEA11, and the genome-wide role of AR in PCOS. We determined that elevated androgen levels on PCOS cells had an impact on the delayed and incomplete decidual transformation of endometrial cells. The AR co-regulator MAGEA11, a known enhancer of AR function, was constitutively overexpressed throughout the menstrual cycle of PCOS patients, co-localised in the nucleus of PCOS stromal tissue and cells and formed a molecular complex with AR. Genome-wide AR analysis in PCOS stromal cells revealed that AR targets included genes involved in cell death and apoptosis, as well as genes commonly dysregulated in endometrial cancer. Enhanced MAGEA11 and AR-mediated transcriptional regulation may impact on a correct endometrial decidualisation response, subsequently affecting endometrial receptivity in these infertile women. KEY MESSAGES: MAGEA11 and AR are overexpressed in hyperandrogenic PCOS patients. MAGEA11-AR overexpression in PCOS correlates with delayed decidualisation. AR and MAGEA11 associate in a molecular complex. AR directly regulates a unique set of genes controlling gene differentiation.

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.

An Abies procera-derived tetracyclic triterpene containing a steroid-like nucleus core and a lactone side chain attenuates in vitro survival of both Fasciola hepatica and Schistosoma mansoni.

Two economically and biomedically important platyhelminth species, Fasciola hepatica (liver fluke) and Schistosoma mansoni (blood fluke), are responsible for the neglected tropical diseases (NTDs) fasciolosis and schistosomiasis. Due to the absence of prophylactic vaccines, these NTDs are principally managed by the single class chemotherapies triclabendazole (F. hepatica) and praziquantel (S. mansoni). Unfortunately, liver fluke resistance to triclabendazole has been widely reported and blood fluke insensitivity/resistance to praziquantel has been observed in both laboratory settings as well as in endemic communities. Therefore, the identification of new anthelmintics is necessary for the sustainable control of these NTDs in both animal and human populations. Here, continuing our work with phytochemicals, we isolated ten triterpenoids from the mature bark of Abies species and assessed their anthelmintic activities against F. hepatica and S. mansoni larval and adult lifecycle stages. Full 1H and 13C NMR-mediated structural elucidation of the two most active triterpenoids revealed that a tetracyclic steroid-like nucleus core and a lactone side chain are associated with the observed anthelmintic effects. When compared to representative mammalian cell lines (MDBK and HepG2), the most potent triterpenoid (700015; anthelmintic EC50s range from 0.7 µM-15.6 µM) displayed anthelmintic selectivity (selectivity indices for F. hepatica: 13 for newly excysted juveniles, 46 for immature flukes, 2 for mature flukes; selectivity indices for S. mansoni: 14 for schistosomula, 9 for immature flukes, 4 for adult males and 3 for adult females) and induced severe disruption of surface membranes in both liver and blood flukes. S. mansoni egg production, a process responsible for pathology in schistosomiasis, was also severely inhibited by 700015. Together, our results describe the structural elucidation of a novel broad acting anthelmintic triterpenoid and support further investigations developing this compound into more potent analogues for the control of both fasciolosis and schistosomiasis.

Methyl-CpG-binding (SmMBD2/3) and chromobox (SmCBX) proteins are required for neoblast proliferation and oviposition in the parasitic blood fluke Schistosoma mansoni.

While schistosomiasis remains a significant health problem in low to middle income countries, it also represents a recently recognised threat to more economically-developed regions. Until a vaccine is developed, this neglected infectious disease is primarily controlled by praziquantel, a drug with a currently unknown mechanism of action. By further elucidating how Schistosoma molecular components cooperate to regulate parasite developmental processes, next generation targets will be identified. Here, we continue our studies on schistosome epigenetic participants and characterise the function of a DNA methylation reader, the Schistosoma mansoni methyl-CpG-binding domain protein (SmMBD2/3). Firstly, we demonstrate that SmMBD2/3 contains amino acid features essential for 5-methyl cytosine (5mC) binding and illustrate that adult schistosome nuclear extracts (females > males) contain this activity. We subsequently show that SmMBD2/3 translocates into nuclear compartments of transfected murine NIH-3T3 fibroblasts and recombinant SmMBD2/3 exhibits 5mC binding activity. Secondly, using a yeast-two hybrid (Y2H) screen, we show that SmMBD2/3 interacts with the chromo shadow domain (CSD) of an epigenetic adaptor, S. mansoni chromobox protein (SmCBX). Moreover, fluorescent in situ hybridisation (FISH) mediated co-localisation of Smmbd2/3 and Smcbx to mesenchymal cells as well as somatic- and reproductive- stem cells confirms the Y2H results and demonstrates that these interacting partners are ubiquitously expressed and found within both differentiated as well as proliferating cells. Finally, using RNA interference, we reveal that depletion of Smmbd2/3 or Smcbx in adult females leads to significant reductions (46-58%) in the number of proliferating somatic stem cells (PSCs or neoblasts) as well as in the quantity of in vitro laid eggs. Collectively, these results further expand upon the schistosome components involved in epigenetic processes and suggest that pharmacological inhibition of SmMBD2/3 and/or SmCBX biology could prove useful in the development of future schistosomiasis control strategies.