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Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP + during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

Schistosomiasis, also known as bilharzia or snail fever, is a tropical parasitic disease resulting from flatworms of the Schistosoma genus. This often overlooked disease has significant impacts in affected regions, causing enduring morbidity, hindering child development, reducing productivity, and creating economic burdens. Praziquantel (PZQ) is currently the only treatment option for schistosomiasis. Given the potential rise of drug resistance and the limited treatment choices available, there is a need to develop more effective inhibitors for this neglected tropical disease (NTD). In view of this, quantitative structure-activity relationship studies (QSAR), molecular docking, molecular dynamics simulations, drug-likeness, and ADMET predictions were applied to 31 inhibitors of Schistosoma mansoni Dihydroorotate dehydrogenase (SmDHODH). The designed QSAR model demonstrated robust statistical parameters including an R2 of 0.911, R2adj of 0.890, Q2cv of 0.686, R2pred of 0.807, and cR2p of 0.825, confirming its robustness. Compound 26, identified as the most active derivative, emerged as a lead candidate for new potential inhibitors through ligand-based drug design. Subsequently, 12 novel compounds (26A-26L) were designed with enhanced inhibition activity and binding affinity. Molecular docking studies revealed strong and stable interactions, including hydrogen bonding and hydrophobic interactions, between the designed compounds and the target receptor. Molecular dynamics simulations over 100 nanoseconds and MM-PBSA free binding energy (ΔGbind) calculations validated the stability of the two best-designed molecules (26A and 26L). Furthermore, drug-likeness and ADMET prediction analyses affirmed the potential of these designed compounds, suggesting their promise as innovative agents for treating schistosomiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ja’afaru et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The NAD + -dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni ( Sm Sirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and Sm Sirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Lactate dehydrogenase (LDH) from Schistosoma mansoni has peculiar properties for a eukaryotic LDH. Schistosomal LDH (SmLDH) isolated from schistosomes, and the recombinantly expressed protein, are strongly inhibited by ATP, which is neutralized by fructose-1,6-bisphosphate (FBP). In the conserved FBP/anion binding site we identified two residues in SmLDH (Val187 and Tyr190) that differ from the conserved residues in LDHs of other eukaryotes, but are identical to conserved residues in FBP-sensitive prokaryotic LDHs. Three-dimensional (3D) models were generated to compare the structure of SmLDH with other LDHs. These models indicated that residues Val187, and especially Tyr190, play a crucial role in the interaction of FBP with the anion pocket of SmLDH. These 3D models of SmLDH are also consistent with a competitive model of SmLDH inhibition in which ATP (inhibitor) and FBP (activator) compete for binding in a well-defined anion pocket. The model of bound ATP predicts a distortion of the nearby key catalytic residue His195, resulting in enzyme inhibition. To investigate a possible physiological role of this allosteric regulation of LDH in schistosomes we made a kinetic model in which the allosteric regulation of the glycolytic enzymes can be varied. The model showed that inhibition of LDH by ATP prevents fermentation to lactate in the free-living stages in water and ensures complete oxidation via the Krebs cycle of the endogenous glycogen reserves. This mechanism of allosteric inhibition by ATP prevents the untimely depletion of these glycogen reserves, the only fuel of the free-living cercariae. Neutralization by FBP of this ATP inhibition of LDH prevents accumulation of glycolytic intermediates when S. mansoni schistosomula are confronted with the sudden large increase in glucose availability upon penetration of the final host. It appears that the LDH of S. mansoni is special and well suited to deal with the variations in glucose availability the parasite encounters during its life cycle., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 ( Sm CB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and Sm CB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs " Sm CB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the " Sm CB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of Sm CB1 ( K i = 0.050 μM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 μM, 68% at 1 μM, 35% at 0.1 μM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species , due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors ( Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.

The emergence of high-throughput methodologies such as next-generation sequencing and proteomics has necessitated significant advancements in biological databases and bioinformatic tools, therefore reshaping the landscape of research into parasitic peptidases. In this review we outline the development of these resources along the -omics technologies and their transformative impact on the field. Apart from extensive summary of general and specific databases and tools, we provide a general pipeline on how to use these resources effectively to identify candidate peptidases from these large datasets and how to gain as much information about them as possible without leaving the office chair. This pipeline is then applied in an illustrative case study on the endothelin-converting enzyme 1 homologue from Schistosoma mansoni and attempts to highlight the contemporary capabilities of bioinformatics. The case study demonstrate how such approach can aid to hypothesize enzyme functions and interactions through computational analysis alone effectively and emphasizes how such virtual investigations can guide and optimize subsequent wet lab experiments therefore potentially saving precious time and resources. Finally, by showing what can be achieved without traditional wet laboratory methods, this review provides a compelling narrative on the use of bioinformatics to bridge the gap between big data and practical research applications, highlighting the key role of these technologies in furthering our understanding of parasitic diseases., (Copyright © 2024. Published by Elsevier Ltd.)

Neglected tropical diseases (NTDs) are prevalent in tropical and subtropical countries, and schistosomiasis is among the most relevant diseases worldwide. In addition, one of the two biggest problems in developing drugs against this disease is related to drug resistance, which promotes the demand to develop new drug candidates for this purpose. Thus, one of the drug targets most explored, Schistosoma mansoni Cathepsin B1 (SmCB1 or Sm31), provides new opportunities in drug development due to its essential functions for the parasite's survival. In this way, here, the latest developments in drug design studies targeting SmCB1 were approached, focusing on the most promising analogs of nitrile, vinyl sulphones, and peptidomimetics. Thus, it was shown that despite being a disease known since ancient times, it remains prevalent throughout the world, with high mortality rates. The therapeutic arsenal of antischistosomal drugs (ASD) consists only of praziquantel, which is widely used for this purpose and has several advantages, such as efficacy and safety. However, it has limitations, such as the impossibility of acting on the immature worm and exploring new targets to overcome these limitations. SmCB1 shows its potential as a cysteine protease with a catalytic triad consisting of Cys 100 , His 270 , and Asn 290 . Thus, design studies of new inhibitors focus on their catalytic mechanism for designing new analogs. In fact, nitrile and sulfonamide analogs show the most significant potential in drug development, showing that these chemical groups can be better exploited in drug discovery against schistosomiasis. We hope this manuscript guides the authors in searching for promising new antischistosomal drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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&#95;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&#95;089290's female biased expression. Spatial localisation of smp&#95;089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp&#95;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&#95;089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp&#95;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., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis is a debilitating parasitic disease caused by intravascular flatworms called schistosomes (blood flukes) that affects >200 million people worldwide. Proteomic analysis has revealed the surprising presence of classical glycolytic enzymes - typically cytosolic proteins - located on the extracellular surface of the parasite tegument (skin). Immunolocalization experiments show that phosphoglycerate mutase (PGM) is widely expressed in parasite tissues and is highly expressed in the tegument. We demonstrate that live Schistosoma mansoni parasites express enzymatically active PGM on their tegumental surface. Suppression of PGM using RNA interference (RNAi) diminishes S. mansoni PGM (SmPGM) gene expression, protein levels, and surface enzyme activity. Sequence comparisons place SmPGM in the cofactor (2,3-bisphosphoglycerate)-dependent PGM (dPGM) family. We have produced recombinant SmPGM (rSmPGM) in an enzymatically active form in Escherichia coli. The Michaelis-Menten constant (K m ) of rSmPGM for its glycolytic substrate (3-phosphoglycerate) is 0.85 mM ± 0.02. rSmPGM activity is inhibited by the dPGM-specific inhibitor vanadate. Here, we show that rSmPGM not only binds to plasminogen but also promotes its conversion to an active form (plasmin) in vitro. This supports the hypothesis that host-interactive tegumental proteins (such as SmPGM), by enhancing plasmin formation, may help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo., (© D.B. Pirovich et al., published by EDP Sciences, 2022.)

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, we chemically optimized our previously reported benzhydroxamate-based inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by the highly potent inhibitor 5o. Structure-based optimization of the novel inhibitors was carried out using the available crystal structures as well as docking studies on smHDAC8. The compounds were evaluated in screens for inhibitory activity against schistosome and human HDACs (hHDAC). The in vitro and docking results were used for detailed structure activity relationships. The synthesized compounds were further investigated for their lethality against the schistosome larval stage using a fluorescence-based assay. The most promising inhibitor 5o showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5' end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5' end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tegumental carbonic anhydrase from the worm Schistosoma mansoni (SmCA) is considered a new anti-parasitic target because suppressing its expression interferes with schistosome metabolism and virulence. Here, we present the inhibition profiles of selenoureido compounds on recombinant SmCA and resolution of the first X-ray crystal structures of SmCA in adduct with a selection of such inhibitors. The key molecular features of such compounds in adduct with SmCA were obtained and compared to the human isoform hCA II, in order to understand the main structural factors responsible for enzymatic affinity and selectivity. Compounds that more specifically inhibited the schistosome versus human enzymes were identified. The results expand current knowledge in the field and pave the way for the development of more potent antiparasitic agents in the near future.

Background: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay., Methodology: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques., Results: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface., Conclusions: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.

The enzyme Glyceraldehyde-3-Phosphate Dehydrogenase from Schistosoma mansoni (SmGAPDH) is characterized as a therapeutical target for schistosomiasis. In this context, we report here the experimental structure, structural analyses and comparisons of SmGAPDH, the first one from a Platyhelminth. The enzyme was expressed, purified and assayed for crystallization, what allowed the obtainment of crystals of sufficient quality to collect X-ray diffraction data up to 2.51 Å resolution. SmGAPDH is the only GAPDH to present the sequence NNR (its residues 114-116) which leads to (especially R116) a hydrogen bond network that possibly reflects on the flexibility of residues to interact with the adenine part of NAD + , speculated to be important for differential drug design., Competing Interests: Declaration of competing interest The authors declare no conflict interests., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Histone-modifying proteins have been identified as promising targets to treat several diseases including cancer and parasitic ailments. In silico methods have been incorporated within a variety of drug discovery programs to facilitate the identification and development of novel lead compounds. In this study, we explore the binding modes of a series of benzhydroxamates derivatives developed as histone deacetylase inhibitors of Schistosoma mansoni histone deacetylase ( sm HDAC) using molecular docking and binding free energy (BFE) calculations. The developed docking protocol was able to correctly reproduce the experimentally established binding modes of resolved sm HDAC8-inhibitor complexes. However, as has been reported in former studies, the obtained docking scores weakly correlate with the experimentally determined activity of the studied inhibitors. Thus, the obtained docking poses were refined and rescored using the Amber software. From the computed protein-inhibitor BFE, different quantitative structure-activity relationship (QSAR) models could be developed and validated using several cross-validation techniques. Some of the generated QSAR models with good correlation could explain up to ~73% variance in activity within the studied training set molecules. The best performing models were subsequently tested on an external test set of newly designed and synthesized analogs. In vitro testing showed a good correlation between the predicted and experimentally observed IC 50 values. Thus, the generated models can be considered as interesting tools for the identification of novel sm HDAC8 inhibitors.

Schistosomiasis is a neglected tropical disease that affects more than 250 million people worldwide. The only drug available for its treatment undergoes first-pass hepatic metabolism and is not capable of preventing reinfection, which makes the search of new therapies urgently needed. Due to the essential role of fumarases in metabolism, these enzymes represent potential targets for developing novel schistosomiasis treatments. Here, we evaluate the expression profiles for class I and class II fumarases from Schistosoma mansoni (SmFH I and SmFH II , respectively), and report the complete characterization of SmFH II . The first SmFH II structure in complex with L-malate was determined at 1.85 Å resolution. The significant thermoshift observed for SmFH II in the presence of identified ligands makes the differential scanning fluorimetry an adequate technique for ligand screening. A complete kinetic characterization of SmFH II was performed, and comparison with the human fumarase (HsFH) revealed differences regarding the turnover number (k cat ). Structural characterization allowed us to identify differences between SmFH II and HsFH that could be explored to design new selective inhibitors. This work represents the very first step towards validate the fumarases as drug targets to treat schistosomiasis. Our results provide the structural basis to rational search for selective ligands., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Da’dara, Elzoheiry, El-Beshbishi and Skelly.)

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4., (© 2020 Federation of European Biochemical Societies.)

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp&#95;154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes., (© 2020 Federation of American Societies for Experimental Biology.)

A previous phenotypic screening campaign led to the identification of a quinazoline derivative with promising in vitro activity against Schistosoma mansoni . Follow-up studies of the antischistosomal potential of this candidate are presented here. The in vivo studies in a S. mansoni mouse model show a significant reduction of total worms and a complete disappearance of immature eggs when administered concomitantly with praziquantel in comparison with the administration of praziquantel alone. This fact is of utmost importance because eggs are responsible for the pathology and transmission of the disease. Subsequently, the chemical optimisation of the structure in order to improve the metabolic stability of the parent compound was carried out leading to derivatives with improved drug-like properties. Additionally, the putative target of this new class of antischistosomal compounds was envisaged by using computational tools and the binding mode to the target enzyme, aldose reductase, was proposed.

Infection with intravascular platyhelminths of the genus Schistosoma can result in the debilitating disease schistosomiasis. Schistosomes (blood flukes) can survive in the host for many years. We hypothesize that proteins on their host-interactive surface modify the worm's external environment to help insure worm survival. Previously, we have shown that a surface ectoenzyme of Schistosoma mansoni , SmNPP5 - a nucleotide pyrophosphatase/phosphodiesterase - can cleave ADP and block platelet aggregation in vitro . In this work, we show that both adult schistosomes and recombinant SmNPP5 can cleave the exogenous purinergic signaling molecule nicotinamide adenine dinucleotide (NAD). In doing so, worms and rSmNPP5 can prevent NAD-induced apoptosis of T cells in vitro . Since regulatory T cells (Tregs) are especially prone to such NAD-induced cell death (NICD), we hypothesize that schistosome cleavage of NAD promotes Treg survival which creates a more immunologically hospitable environment for the worms in vivo . In addition to SmNPP5, schistosomes express another host-interactive NAD-degrading enzyme, SmNACE. We successfully suppressed the expression of SmNPP5 and SmNACE (singly or together) using RNAi. Only SmNPP5-suppressed worms, and not SmNACE-suppressed worms, were significantly impaired in their ability to cleave exogenous NAD compared to controls. Therefore, we contend that ectoenzyme SmNPP5 on the surface of the worm is primarily responsible for extracellular NAD cleavage and that this helps modulate the host immune environment by preventing Treg cell death.

Schistosomiasis threatens 800 million people worldwide. Chronic pathology manifests as hepatosplenomegaly, and intestinal schistosomiasis caused by Schistosoma mansoni can lead to liver fibrosis, cirrhosis, and blood in the stool. To assist the only FDA-approved drug, praziquantel, in parasite elimination, the development of a vaccine would be of high value. S. mansoni Cathepsin B (SmCB) is a well-documented vaccine target for intestinal schistosomiasis. Herein, we test the increased efficacy and immunogenicity of SmCB when combined with sulfated lactosyl archaeol (SLA) archaeosomes or AddaVax™ (a squalene based oil-in-water emulsion). Both vaccine formulations resulted in robust humoral and cell mediated immune responses. Impressively, both formulations were able to reduce parasite burden greater than 40% (WHO standard), with AddaVax™ reaching 86.8%. Additionally, SmCB with both adjuvants were able to reduce granuloma size and the amount of larval parasite hatched from feces, which would reduce transmission. Our data support SmCB as a target for S. mansoni vaccination; especially when used in an adjuvanted formulation., (Copyright © 2020 Perera, Hassan, Jia, Ricciardi, McCluskie, Weeratna and Ndao.)

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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni , Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni . On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease., (Copyright © 2020 Cagnazzo, Nogueira, Castro, Neris, Fattori, Correia, Albuquerque, Fragelli, Mendes, Allegretti, Soares, Romanello, Torini, Pereira and Anibal.)

Background: Proteins are efficient supramolecular scaffolds to drive self-assembly of nanomaterials into regular colloidal structures suitable for several purposes, including cell imaging and drug delivery. Proteins, in particular, can bind to gold nanoparticles (AuNPs) through van der Waals and electrostatic forces as well as coordination and hydrogen bonds leading their assembly into responsive nanostructures., Methods: Bioconjugation of alkyne Raman tag-labeled 20 nm AuNPs with the ring-shaped protein Peroxiredoxin (Prx), characterized by a symmetric homo-oligomeric circular arrangement, has been investigated by absorption spectroscopy, transmission and scanning electron microscopy. The plasmonic behavior of the resulting hybrid assemblies has been assessed by Surface Enhanced Raman Scattering (SERS)., Results: The ring-shaped Prx molecules are demonstrated to adsorb onto the gold surface acting as "sticky" bio-linkers between adjacent nanoparticles to drive self-assembly into small colloidal AuNPs arrays. The arrays show nanometric interparticle gaps tailored by the protein ring thickness. The arrays exhibit improved optical activity due to SERS allowing detection of the Raman signals from both the protein and alkyne molecules., Conclusions: This method can be used to build up SERS-active nanostructures using Prx as both a bio-linker and platform for attaching dyes, two-dimensional materials, such as graphene, and other biomolecules including DNA and enzymes., General Significance: The development of colloidal SERS nanostructures is considered a significant step forward in spectroscopic bioanalysis. Though protein-tailored nanofabrication is in a childhood stage, these results demonstrate the versatility of supramolecular proteins as tools to build-up nanostructures which are still impractical to obtain through top-down techniques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, which affects over 200 million people worldwide and leads to at least 300,000 deaths every year. In this study, initial screening revealed the triazole-based hydroxamate 2 b (N-hydroxy-1-phenyl-1H-1,2,3-triazole-4-carboxamide) exhibiting potent inhibitory activity toward the novel antiparasitic target Schistosoma mansoni histone deacetylase 8 (smHDAC8) and promising selectivity over the major human HDACs. Subsequent crystallographic studies of the 2 b/smHDAC8 complex revealed key interactions between the inhibitor and the enzyme's active site, thus explaining the unique selectivity profile of the inhibitor. Further chemical modifications of 2 b led to the discovery of 4-fluorophenoxy derivative 21 (1-[5-chloro-2-(4-fluorophenoxy)phenyl]-N-hydroxy-1H-1,2,3-triazole-4-carboxamide), a nanomolar smHDAC8 inhibitor (IC 50 =0.5 μM), exceeding the smHDAC8 inhibitory activity of 2 b and SAHA (vorinostat), while exhibiting an improved selectivity profile over the investigated human HDACs. Collectively, this study reveals specific interactions between smHDAC8 and the synthesized triazole-based inhibitors and demonstrates that these small molecules represent promising lead structures, which could be further developed in the search for novel drugs for the treatment of schistosomiasis., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Schistosomes are intravascular blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic analysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis-Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thrombolysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of Schistosoma mansoni TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against ex vivo worms. Compounds 1 - 5 are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds 1 - 5 , 7 , and 8 displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds 1 - 4 meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds 4 - 6 were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC 50 of 5 µM and a K d of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC 50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC 50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health.

Triosephosphate isomerases (TPIs) from Taenia solium (TsTPI) and Schistosoma mansoni (SmTPI) are potential vaccine and drug targets against cysticercosis and schistosomiasis, respectively. This is due to the dependence of parasitic helminths on glycolysis and because those proteins elicit an immune response, presumably due to their surface localization. Here we report the crystal structures of TsTPI and SmTPI in complex with 2-phosphoglyceric acid (2-PGA). Both TPIs fold into a dimeric (β-α)8 barrel in which the dimer interface consists of α-helices 2, 3, and 4, and swapping of loop 3. TPIs from parasitic helminths harbor a region of three amino acids knows as the SXD/E insert (S155 to E157 and S157 to D159 in TsTPI and SmTPI, respectively). This insert is located between α5 and β6 and is proposed to be the main TPI epitope. This region is part of a solvent-exposed 310-helix that folds into a hook-like structure. The crystal structures of TsTPI and SmTPI predicted conformational epitopes that could be used for vaccine design. Surprisingly, the epitopes corresponding to the SXD/E inserts are not the ones with the greatest immunological potential. SmTPI, but not TsTPI, habors a sole solvent exposed cysteine (SmTPI-S230) and alterations in this residue decrease catalysis. The latter suggests that thiol-conjugating agents could be used to target SmTPI. In sum, the crystal structures of SmTPI and TsTPI are a blueprint for targeted schistosomiasis and cysticercosis drug and vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Schistosomiasis (also known as bilharzia) is a neglected tropical disease caused by platyhelminths of the genus Schistosoma . The disease is endemic in tropical and subtropical areas of the world where water is infested by the intermediate parasite host, the snail. More than 800 million people live in endemic areas and more than 200 million are infected and require treatment. Praziquantel (PZQ) is the drug of choice for schistosomiasis treatment and transmission control being safe and very effective against adult worms of all the clinically relevant Schistosoma species. Unfortunately, it is ineffective on immature, juvenile worms; therefore, it does not prevent reinfection. Moreover, the risk of development and spread of drug resistance because of the widespread use of a single drug in such a large population represents a serious threat. Therefore, research aimed at identifying novel drugs to be used alone or in combination with PZQ are needed. Schistosoma mansoni histone deacetylase 8 ( Sm HDAC8) is a class I zinc-dependent HDAC, which is abundantly expressed in all stages of its life cycle, thus representing an interesting target for drug discovery. Through virtual screening and phenotypical characterization of selected hits, we discovered two main chemical classes of compounds characterized by the presence of a hydroxamate-based metal binding group coupled to a spiroindoline or a tricyclic thieno[3,2- b ]indole core as capping groups. Some of the compounds of both classes were deeply investigated and showed to impair viability of larval, juvenile, and adult schistosomes, to impact egg production in vitro and/or to induce morphological alterations of the adult schistosome reproductive systems. Noteworthy, all of them inhibit the recombinant form of Sm HDAC8 enzyme in vitro . Overall, we identified very interesting scaffolds, paving the way to the development of effective antischistosomal agents.

Dihydrofolate reductase (DHFR) is an essential enzyme for nucleotide metabolism used to obtain energy and structural nucleic acids. Schistosoma mansoni has all the pathways for pyrimidine biosynthesis, which include the thymidylate cycle and, consequentially, the DHFR enzyme. Here, we describe the characterization of Schistosoma mansoni DHFR (SmDHFR) using isothermal titration calorimetry for the enzymatic activity and thermodynamic determination, also the folate analogs inhibition. Moreover, X-ray crystallography was used to determine the enzyme atomic model at 1.95 Å.

Praziquantel (PZQ) is the sole drug used to treat schistosomiasis, and the probability of developing resistance is growing the longer it is relied upon, justifying the search for alternatives. Phosphodiesterases (PDEs), particularly the PDE4 family, have attracted considerable attention as drug targets, including in Schistosoma mansoni, and especially SmPDE4A. This study investigates the potential antischistosomal activity of human PDE4 and potent SmPDE4A inhibitor roflumilast, either alone or combined with PZQ. In vitro, roflumilast resulted in a significant, concentration-dependent reduction in egg production but not of worm viability. In vitro exposure to roflumilast in combination with a low concentration of PZQ was less effective than PZQ alone, pointing to antagonism. S. mansoni-infected mice treated with roflumilast showed significant reductions in worm burden (27%) as well as hepatic and intestinal egg burdens (~28%) two weeks post treatment. Scanning EM of worms isolated from roflumilast-treated and untreated mice did not reveal noticeable changes to their tegument. S. mansoni-infected mice treated with a fixed dosage of roflumilast and a variable dosage of PZQ resulted in a higher reduction in worm burden, reduced hepatic egg counts, absence of immature eggs and a marked increase in dead eggs, compared to PZQ alone. However, the combination resulted in increased animal mortality, probably attributable to pharmacodynamic interactions between the two drugs. Although this study marks the first report of in vivo antischistosomal potential by a PDE inhibitor, an important proof of concept, we conclude that the antischistosomal effects of roflumilast are insufficient to warrant further development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein structure determination by X-ray crystallography guides structure-function and rational drug design studies. Helminths cause devastating diseases, including schistosomiasis that affects over one-third of the human population. Trematodes from the genus Schistosoma heavily depend on glycolysis; thus enzymes involved in this metabolic pathway are potential drug targets. Here we present a protocol to obtain crystal structures of recombinantly expressed triosephosphate isomerase from S. mansoni (SmTPI) that diffracted in house to a resolution of 2 Å.

Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp&#95;154600 and Smache2 -smp&#95;136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp&#95;125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP., Competing Interests: The authors have declared that no competing interests exist

Schistosoma mansoni is a long-lived intravascular trematode parasite that can infect humans causing the chronic debilitating disease, schistosomiasis. We hypothesize that the action of host-interactive proteins found at the schistosome surface allows the worms to maintain a safe, anti-thrombotic and anti-inflammatory environment around them in the bloodstream. One such protein is the ˜60 kDa alkaline phosphatase SmAP which is known to be expressed in the outer tegument of all intravascular life stages. We demonstrate in this work that the parasites (schistosomula as well as adult males and females) can hydrolyze polyphosphate (polyP) - an anionic, linear polymer of inorganic phosphates that is produced and released by immune cells as well as by activated platelets and that induce proinflammatory and prothrombotic pathways. Purified recombinant SmAP can likewise cleave polyP and with a K m of 6.9 ± 1 mM. Finally, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to hydrolyze polyP. SmAP-mediated cleavage of polyP may contribute to the armamentarium of schistosomes that promotes their survival in the hostile intravascular habitat. This is the first report of any pathogen cleaving this bioactive metabolite., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [ 14 C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no 14 CO 2 production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Aim: Due to the urgent need for effective drugs to treat schistosomiasis that act through a known molecular mechanism of action, we focused on a target-based approach with the aim to discover inhibitors of a cyclic nucleotide phosphodiesterase from Schistosoma mansoni (SmPDE4A). Materials & methods: To discover new inhibitors of SmPDE4A homology models of the enzyme structure were constructed based on known human and protozoan homologs. The best two models were selected for subsequent virtual screening of our in-house chemical library. Results & conclusion: A total of 25 library compounds were selected for experimental confirmation as SmPDE4A inhibitors and after dose-response experiments, three top hits were identified. The results presented validate the virtual screening approach to identify new inhibitors for clinically relevant phosphodiesterases.

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Schistosomiasis ranks second only to malaria as the most common parasitic disease worldwide. 700 million people are at risk and 240 million are already infected. Praziquantel is the anthelmintic of choice but decreasing efficacy has already been documented. In this work, we exploited the inhibition of Schistosoma mansoni dihydroorotate dehydrogenase (SmDHODH) as a strategy to develop new therapeutics to fight schistosomiasis. A series of quinones (atovaquone derivatives and precursors) was evaluated regarding potency and selectivity against both SmDHODH and human DHODH. The best compound identified is 17 (2-hydroxy-3-isopentylnaphthalene-1,4-dione) with IC 50  = 23 ± 4 nM and selectivity index of 30.83. Some of the new compounds are useful pharmacological tools and represent new lead structures for further optimization., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target., (Copyright © 2019 Elsevier B.V. All rights reserved.)