Search

Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent anti-trypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments.

Schistosomiasis is a chronic and painful disease of poverty caused by the flatworm parasite Schistosoma. Drug discovery for antischistosomal compounds predominantly employs in vitro whole organism (phenotypic) screens against two developmental stages of Schistosoma mansoni, post-infective larvae (somules) and adults. We generated two rule books and associated scoring systems to normalize 3898 phenotypic data points to enable machine learning. The data were used to generate eight Bayesian machine learning models with the Assay Central software according to parasite's developmental stage and experimental time point (≤24, 48, 72, and >72 h). The models helped predict 56 active and nonactive compounds from commercial compound libraries for testing. When these were screened against S. mansoni in vitro, the prediction accuracy for active and inactives was 61% and 56% for somules and adults, respectively; also, hit rates were 48% and 34%, respectively, far exceeding the typical 1-2% hit rate for traditional high throughput screens.

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

The eggs of the parasitic blood fluke, Schistosoma, are the main drivers of the chronic pathologies associated with schistosomiasis, a disease of poverty afflicting approximately 220 million people worldwide. Eggs laid by Schistosoma mansoni in the bloodstream of the host are encapsulated by vascular endothelial cells (VECs), the first step in the migration of the egg from the blood stream into the lumen of the gut and eventual exit from the body. The biomechanics associated with encapsulation and extravasation of the egg are poorly understood. We demonstrate that S. mansoni eggs induce VECs to form two types of membrane extensions during encapsulation; filopodia that probe eggshell surfaces and intercellular nanotubes that presumably facilitate VEC communication. Encapsulation efficiency, the number of filopodia and intercellular nanotubes, and the length of these structures depend on the egg’s vitality and, to a lesser degree, its maturation state. During encapsulation, live eggs induce VEC contractility and membranous structures formation, in a Rho/ROCK pathway-dependent manner. Using elastic hydrogels embedded with fluorescent microbeads as substrates to culture VECs, live eggs induce VECs to exert significantly greater contractile forces during encapsulation than dead eggs, which leads to 3D deformations on both the VEC monolayer and the flexible substrate underneath. These significant mechanical deformations cause the VEC monolayer tension to fluctuate with eventual rupture of VEC junctions, thus facilitating egg transit out of the blood vessel. Overall, our data on the mechanical interplay between host VECs and the schistosome egg improve our understanding of how this parasite manipulates its immediate environment to maintain disease transmission.

Rhodesain is the generic name for the cathepsin L-like peptidase of Trypanosoma brucei rhodesiense. The term rhodesain was derived from the subspecies epithet rhodesiense which itself originated form Rhodesia, a historical region in southern Africa named after the 19th century British imperialist and white supremacist Cecil Rhodes. This tainting could be grounds for discontinuing the name, however, there are also scientific grounds. Specifically, protein sequence comparisons and frequency-based difference profiling reveal that rhodesain is essentially identical (99.87-98.44%) to the cathepsin L-like peptidases of both T. b. brucei and T. b. gambiense. Accordingly, and based on a previously proposed terminology for kinetoplastid C1 peptidases (Caffrey and Steverding, 2009), we suggest the use of the formal term, TbrCATL, to denote the cathepsin L-like peptidases of the T. brucei subspecies. The earlier and informal term, 'brucipain', could also be used.

We recently reported a series of compounds for a solubility-driven optimization campaign of antitrypanosomal compounds. Extending a parasite-hopping approach to the series, a subset of compounds from this library has been cross-screened for activity against the metazoan flatworm parasite, Schistosoma mansoni. This study reports the identification and preliminary development of several potently bioactive compounds against adult schistosomes, one or more of which represent promising leads for further assessment and optimization.

Background Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. Methods Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. Results For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearsonʼs correlations (0.48–0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. Conclusions The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified.

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

Background Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1). Results Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host–parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion. Conclusions SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Schistosomiasis is a parasitic disease that affects approximately 200 million people in developing countries. Current treatment relies on just one partially effective drug and new drugs are needed. Tubulin and microtubules (MTs) are essential constituents of the cytoskeleton in all eukaryotic cells and considered potential drug targets to treat parasitic infections. The α- and β-tubulin of Schistosoma mansoni have ~96% and ~91% sequence identity to their respective human tubulins suggesting that compounds which bind mammalian tubulin may interfere with MT-mediated functions in the parasite. To explore the potential of different classes of tubulin-binding molecules as anti-schistosomal leads, we completed a series of in vitro whole-organism screens of a target-based compound library against S. mansoni adults and somules (post-infective larvae), and identified multiple biologically active compounds, among which phenylpyrimidines were the most promising. Further structure-activity relationship studies of these hits identified a series of novel thiophen-2-yl-pyrimidine congeners, which induce a potent and long-lasting paralysis of the parasite. Moreover, compared to the originating compounds, which showed cytotoxicity values in the low nanomolar range, these new derivatives were 1 – 4 orders of magnitude less cytotoxic and exhibited weak or undetectable activity against mammalian MTs in a cell-based assay of MT stabilization. Given their selective anti-schistosomal activity and relatively simple drug-like structures, these molecules hold promise as candidates for the development of new treatments for schistosomiasis.

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., Graphical abstract Image 1, Highlights • Lead compound 22 was identified with EC50 of 0.44 µM and 84.7 nM for schistosomula and adult worms. • 20 analogues of the lead compound 22 were synthesised. • Compounds 25, 30 and 32 showed the best selectivity profile. • Compounds 31 and 33 are the most active on three medically important schistosome species.

Approximately 10% of the world’s population is at risk of schistosomiasis, a disease of poverty caused by the Schistosoma parasite. To facilitate drug discovery for this complex flatworm, we developed an automated high-content screen to quantify the multidimensional responses of Schistosoma mansoni post-infective larvae (somules) to chemical insult. We describe an integrated platform to process worms at scale, collect time-lapsed, bright-field images, segment highly variable and touching worms, and then store, visualize, and query dynamic phenotypes. To demonstrate the methodology, we treated somules with seven drugs that generated diverse responses and evaluated 45 static and kinetic response descriptors relative to concentration and time. For compound screening, we used the Mahalanobis distance to compare multidimensional phenotypic effects induced by 1323 approved drugs. Overall, we characterize both known anti-schistosomals and identify new bioactives. Apart from facilitating drug discovery, the multidimensional quantification provided by this platform will allow mapping of chemistry to phenotype., Steven Chen et al. develop an automated, time-lapsed, high-content screen to quantify the responses of Schistosoma mansoni larvae to chemical insult. They apply their method to evaluate 45 static and kinetic response endpoints for seven drugs and screen 1323 approved drugs. Their work identifies anti-schistosomal compounds and underscores the value of quantifying motion in phenotypic drug discovery.

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

Schistosome biology illuminated Schistosomiasis is caused by a parasitic flatworm about which little is known. Therefore, options to combat human disease caused by schistosome infection are limited. To aid in our quest to develop treatments, two studies undertook molecular investigations of the parasite Schistosoma mansoni . By generating a single-cell atlas, Wendt et al. identified the developmental trajectory of the flatworm, including the blood-feeding gut required for its survival in the host. From these data, they found a gene required for gut development that, when knocked out through RNA interference, confers reduced pathology in infected mice. Wang et al. performed a large-scale RNA interference survey of S. mansoni and identified an essential pair of protein kinases that can be targeted by approved pharmacological intervention (see the Perspective by Anderson and Duraisingh). These molecular investigations add to our understanding of the schistosome parasite and provide biological information that may help to combat this neglected tropical disease. Science , this issue p. 1644 , p. 1649 ; see also p. 1562

The chemotherapy of schistosomiasis remains centered in the use of praziquantel, however, there has been growing resistant parasites to this drug. Thus, the aim of this work was to evaluate in vitro schistosomicidal activity of the hexanes/dichloromethane 1 : 1 extract of Brazilian green propolis (Pex), as well as its major isolated compounds artepillin C, caffeic acid, coumaric acid and drupanin against Schistosoma mansoni. The Pex was active by displaying an IC50 value of 36.60 (26.26-51.13) μg mL-1 at 72 h against adult worms of S. mansoni. The major isolated compounds were inactive with IC50 values >100 μM, however, the combination of the isolated compounds (CM) in the same range found in the extract was active with an IC50 value of 41.17 (39.89-42.46) μg mL-1 at 72 h. Pex and CM induced alteration in the tegument of S. mansoni, and caffeic acid caused alteration in egg's maturation. Pex displayed in vitro activity against adult worms' and eggs' viability of S. mansoni, which opens new perspectives to better understand the synergistic and/or additive effects promoted by both Pex extract and CM against schistosomiasis features.

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery., Screening for new anthelmintic compounds that are active against parasitic nematodes is costly and labour intensive. Here, the authors use the non-parasitic nematode Caenorhabditis elegans to identify 30 anthelmintic lead compounds in an effective and cost-efficient manner.

Schistosomiasis is an ancient and chronic neglected tropical disease that infects over 240 million people and kills over 200,000 of the world’s poorest people every year1, 2. There are no vaccines and because there is only one drug available, the need for new therapeutics is great. The causative agents of this disease are flatworm parasites that dwell inside the host’s circulation, often for decades, where they feed on blood and lay eggs which are primarily responsible for disease pathology. As metazoans comprised of multiple tissue types, understanding the schistosome’s tissues on a molecular level and their functions during what can be decades of successful parasitism could suggest novel therapeutic strategies. Here, we employ single-cell RNAseq to characterize 43,642 cells from the pathogenic (adult) stage of the schistosome lifecycle. From these data, we characterize 68 molecularly distinct cell populations that comprise nearly all tissues described morphologically, including the nervous and reproductive systems. We further uncover a lineage of somatic stem cells responsible for producing and maintaining the parasite’s gut – the primary tissue responsible for digestion of host blood. Finally, we show that a homologue of hepatocyte nuclear factor 4 (hnf4) is expressed in this gut lineage and required for gut maintenance, blood feeding and inducing egg-associated pathology in vivo. Together, the data highlight the utility of this single-cell RNAseq atlas to understand schistosome biology and identify potential therapeutic interventions.

The neglected tropical disease, schistosomiasis, is caused by trematode blood flukes of the Schistosoma genus and infects approximately 200 million people worldwide. With just one partially effective drug available for disease treatment, new drugs are urgently needed. Herein, a series of 47 phthalimide (Pht) analogues possessing high-value bioactive scaffolds (i.e., benzimidazole and 1,2,3,-triazoles) was synthesized by click-chemistry. Compounds were evaluated for anti-schistosomal activity in culture against somules (post-infective larvae) and adults of Schistosoma mansoni, their predicted ADME (absorption, distribution, metabolism, and excretion) properties, and toxicity vs. HepG2 cells. The majority showed favorable parameters for surface area, lipophilicity, bioavailability and Lipinski score. Thirteen compounds were active at 10 &micro, M against both somules and adults (6d, 6f, 6i&ndash, 6l, 6n&ndash, 6p, 6s, 6r&rsquo, 6t&rsquo, and 6w). Against somules, the majority caused degeneracy and/or death after 72 h, whereas against adult parasites, five compounds (6l, 6d, 6f, 6r&rsquo, and 6s) elicited degeneracy, tegumental (surface) damage and/or death. Strongest potency against both developmental stages was recorded for compounds possessing n-butyl or isobutyl as a linker, and a pentafluorophenyl group on triazole. Apart from five compounds for which anti-parasite activity tracked with toxicity to HepG2 cells, there was apparently no toxicity to HepG2 cells (EC50 values &ge, 50 &micro, M). The data overall suggest that phthaloyl-triazole compounds are favorable synthons for additional studies as anti-schistosomals.

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 IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 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 187in 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.

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target., Graphical abstract Image 1, Highlights • TPT sulfonate provides single dose efficacy that is competitive with the current drug, praziquantel. • TPT sulfonate must be biotransformed to be active. • TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism. • One of the key metabolites, TP thiol, is anti-schistosomal in vitro and in vivo.

Schistosomiasis is a disease caused by a flatworm parasite that infects people in tropical and subtropical regions of Sub-Saharan Africa, South America, China, and Southeast Asia. The reliance on just one drug for current treatment emphasizes the need for new chemotherapeutic strategies. The aim of this study was to determine the phenotypic effects of extracts and fractions of leaf and stem bark of Erythrophleum ivorense (family Euphorbiaceae), a tree that grows in tropical parts of Africa, on two developmental stages of Schistosoma mansoni, namely, postinfective larvae (schistosomula or somules) and adults. Methanol leaf and stem bark extracts of E. ivorense were successively fractionated with acetone, petroleum ether, ethyl acetate, and methanol. These fractions were then incubated with somules at 0.3125 to 100 μg/mL and with adults at 1.25 μg/mL. The acetone fractions of both the methanol leaf and bark of E. ivorense were most active against the somules whereas the petroleum ether fractions showed least activity. For adult parasites, the acetone fraction of methanol bark extract also elicited phenotypic changes. The data arising provide the first step in the discovery of new treatments for an endemic infectious disease using locally sourced African medicinal plants.

Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Amongst these, schistosomiasis (bilharzia or ‘snail fever’), caused by blood flukes of the genus Schistosoma, ranks second only to malaria in terms of human impact: two hundred million people are infected and close to 800 million are at risk of infection. Drug screening against helminths poses unique challenges: the parasite cannot be cloned and is difficult to target using gene knockouts or RNAi. Consequently, both lead identification and validation involve phenotypic screening, where parasites are exposed to compounds whose effects are determined through the analysis of the ensuing phenotypic responses. The efficacy of leads thus identified derives from one or more or even unknown molecular mechanisms of action. The two most immediate and significant challenges that confront the state-of-the-art in this area are: the development of automated and quantitative phenotypic screening techniques and the mapping and quantitative characterization of the totality of phenotypic responses of the parasite. In this paper, we investigate and propose solutions for the latter problem in terms of the following: (1) mathematical formulation and algorithms that allow rigorous representation of the phenotypic response space of the parasite, (2) application of graph-theoretic and network analysis techniques for quantitative modeling and characterization of the phenotypic space, and (3) application of the aforementioned methodology to analyze the phenotypic space of S. mansoni – one of the etiological agents of schistosomiasis, induced by compounds that target its polo-like kinase 1 (PLK 1) gene – a recently validated drug target. In our approach, first, bio-image analysis algorithms are used to quantify the phenotypic responses of different drugs. Next, these responses are linearly mapped into a low- dimensional space using Principle Component Analysis (PCA). The phenotype space is modeled using neighborhood graphs which are used to represent the similarity amongst the phenotypes. These graphs are characterized and explored using network analysis algorithms. We present a number of results related to both the nature of the phenotypic space of the S. mansoni parasite as well as algorithmic issues encountered in constructing and analyzing the phenotypic-response space. In particular, the phenotype distribution of the parasite was found to have a distinct shape and topology. We have also quantitatively characterized the phenotypic space by varying critical model parameters. Finally, these maps of the phenotype space allows visualization and reasoning about complex relationships between putative drugs and their system-wide effects and can serve as a highly efficient paradigm for assimilating and unifying information from phenotypic screens both during lead identification and lead optimization.

Entamoeba histolytica and Giardia lamblia are parasitic protozoa that cause gastrointestinal disorders such as diarrhea and dysentery. Naegleria fowleri is a free living amoeba that causes primary amoebic meningoencephalitis. However, there are limited treatments for these parasitic diseases. Extracts, fractions from extracts and some isolated compounds from selected Ghanaian medicinal plants were screened against Naegleria fowleri, Giardia lamblia and Entamoeba histolytica in the search for newer and safer agents for the treatment of infections caused by these parasites. Of all the extracts and compounds tested for the activity against E. histolytica, only xylopic acid and geraniin were active with IC50 values of 4.80 μg/mL (13.30 μM) and 34.71 μg/mL (36.44 μM), respectively. Metronidazole, the positive control had an IC50 of 1.287 μM. All other extracts and fractions exhibited IC50 values >100 µg/mL. For G. lamblia, extracts of Albizia glaberrima, Margaritaria nobilis, Maerua angolensis and Ulva fasciata, the ethyl acetate fraction of Erythrophleum ivorense bark extract, and the isolated compound, xylopic acid exhibited IC50 values of 15.91, 44.25, 20.00, 35.86, 13.76 and 11.45 µg/mL, respectively. The IC50 of the positive control agent metronidazole, was 10.47 µM. The extract of A. glaberrima and xylopic acid exhibited IC50 values of 38.70 and 16.06 µg/mL, respectively, against N. fowleri. The IC50 of the reference drug, amphotericin B, was 0.2 µM. Thus, Ghanaian medicinal plant extracts, their fractions and isolated compounds possess anti-parasitic activity. Key words: Naegleria fowleri, Giardia lamblia, Entamoeba histolytica, medicinal plants, geraniin, xylopic acid, Albizia glaberrima, Margaritaria nobilis, Maerua angolensis, Ulva fasciata

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents.

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 μM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 μM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs. A doença de Chagas, a tripanossomíase humana africana e a esquistossomose são doenças parasitárias negligenciadas para as quais são urgentemente necessários novos tratamentos. Para identificar novas pistas químicas, selecionamos os 400 compostos do Open Access Malaria Box contra as proteases de cisteína, cruzaína (Trypanosoma cruzi), rodesaína (Trypanosoma brucei) e SmCB1 (Schistosoma mansoni), que são alvos terapêuticos para essas doenças. Enquanto apenas três acertos foram observados para SmCB1, 70 compostos inibiram cruzaína ou rodesaína em pelo menos 50% a 5μM. Entre esses, 15 compostos disponíveis comercialmente foram selecionados para ensaios confirmatórios, devido à sua potência, perfil de inibição dependente do tempo e atividade relatada contra parasitas. Ensaios adicionais levaram à confirmação de quatro novas classes de inibidores de cruzaína e rodesaína, com potência na faixa de micromolar baixa a média contra enzimas e T. cruzi. Ensaios contra catepsinas S e B de mamíferos revelaram seletividade de inibidores para proteases parasitárias. Para os dois inibidores competitivos identificados (compostos 7 e 12), seu modo de ligação foi previsto por docking, fornecendo uma base para esforços de otimização baseados em estrutura. O composto 12 também agiu diretamente contra as formas tripomastigotas e amastigotas intracelulares de T. cruzi a 3μM. Portanto, através de uma combinação de abordagens experimentais e computacionais, relatamos acertos promissores para otimização no desenvolvimento de novas drogas tripanocidas.

The protozoan parasite Entamoeba histolytica can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the Schistosoma flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both E. histolytica and Schistosoma mansoni genomes encode FT genes. In this study, we phenotypically screened E. histolytica and S. mansoni in vitro with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of Trypanosoma brucei and Trypanosoma cruzi. For E. histolytica, we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 μM against E. histolytica. Apart from five analogs of tipifarnib exhibiting activity against both E. histolytica and S. mansoni, 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 μM after 24 h). Analysis of the structure-activity relationship available for the T. brucei FT suggests that FT may not be the relevant target in E. histolytica and S. mansoni. For E. histolytica, combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis.

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven β subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the β1 and β5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.

New treatments are needed for neglected tropical diseases (NTDs) such as Human African trypanosomiasis (HAT), Chagas disease, and schistosomiasis. Through a whole organism high-throughput screening campaign, we previously identified 797 human kinase inhibitors that grouped into 59 structural clusters and showed activity against T. brucei, the causative agent of HAT. We herein report the results of further investigation of one of these clusters consisting of substituted isatin derivatives, focusing on establishing structure-activity and -property relationship scope. We also describe their in vitro absorption, distribution, metabolism, and excretion (ADME) properties. For one isatin, NEU-4391, which offered the best activity-property profile, pharmacokinetic parameters were measured in mice., The authors wish to acknowledge funding from the National Institute of Allergy and Infectious Diseases (MPP & MN: R01AI114685; MPP R21AI126296, R21AI127594; CRC: R21AI126296, R21AI133393. https://www.niaid.nih.gov/), the Spanish Ministerio de Economía, Industria y Competitividad, (MN: SAF2015-71444-P; DG-P: SAF2016-79957-R. http://www.mineco.gob.es), Junta de Andalucía (FG: CTS-7282. https://www.juntadeandalucia.es) and Subdirección General de Redes y Centros de Investigación Cooperativa (RICET) (MN: RD16/0027/0019; DG-P: RD16/0027/0014. https://bit.ly/2OegOnX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Hookworms remain a major health burden in the developing world, with hundreds of millions currently afflicted by these blood-feeding parasites. There exists a vital need for the discovery of novel drugs and identification of parasite drug targets for the development of chemotherapies. New drug development requires the identification of compounds that target molecules essential to parasite survival and preclinical testing in robust, standardized animal models of human disease. This process can prove costly and time consuming using conventional, low-throughput methods. We have developed a novel high-throughput screen (HTS) to identify anthelmintics for the treatment of soil-transmitted helminths. Our high-throughput, plate reader-based assay was used to rapidly assess compound toxicity to Ancylostoma ceylanicum L1 larva. Using this method, we screened 39,568 compounds from several small molecule screening libraries at 10 μM and identified 830 bioactive compounds that inhibit egg hatching of the human hookworm A. ceylanicum by >50%. Of these, 132 compounds inhibited hookworm egg hatching by >90% compared to controls. The nematicidal activities of 268 compounds were verified by retesting in the egg hatching assay and were also tested for toxicity against the human HeLa cell line at 10 μM. Fifty-nine compounds were verified to inhibit A. ceylanicum egg hatching by >80% and were

The natural history of schistosome infection in the mammalian host is determined by CD4+ T helper responses mounted against different parasite life cycle stages. A T helper 2 (TH2) response to schistosome eggs is required for host survival and establishment of chronic infection. However, a TH2 cell-derived cytokine also contributes to an immune milieu that is conducive to schistosome growth and development. Thus, the same responses that allow for host survival have been co-opted by schistosomes to facilitate parasite development and transmission, underscoring the significance of CD4+ T cell responses to both worms and eggs in the natural history of schistosome infection. Here we show that a cathepsin B1 cysteine protease secreted by schistosome worms not only induces TH2 responses, but also TH1 and TH17 responses, by a mechanism that is dependent on the proteolytic activity of the enzyme. Further investigation revealed that, in addition to the expected TH1 and TH2 responses, acute schistosome infection also induces a transient TH17 response that is rapidly down-regulated at the onset of oviposition. TH17 responses are implicated in the development of severe egg-induced pathology. The regulation of worm-induced TH17 responses during acute infection could therefore influence the expression of high and low pathology states as infection progresses., Author summary Schistosomiasis, a neglected tropical disease caused by parasites of the genus Schistosoma, is prevalent throughout the developing world, with more than 230 million people infected. Left untreated, schistosome infection may cause relatively mild disease with some morbidity, or, in a minority of cases, result in severe pathology and death. These variable outcomes are recapitulated in animal models, where the natural history of schistosome infection is profoundly influenced by the responses of host CD4+ T helper cells. Type 2 CD4+ T cell (TH2) responses, which allow for host survival by limiting pathology, have ironically also been co-opted by schistosomes to promote parasite development. On the other hand, TH17 responses have been implicated in the development of severe pathology, in both experimentally infected animals and naturally infected humans. Here we show that a schistosome proteolytic enzyme (SmCB1), produced in the parasite gut and released into the bloodstream, induces both TH2 and TH17 responses by a mechanism that requires the enzyme’s inherent proteolytic activity. Further investigation revealed that acute schistosome infection also induces a transient TH17 response that is rapidly down-regulated once parasite egg-laying commences. Regulation of TH17 responses during early infection may help determine whether mild or severe pathology develops as the infection progresses.

Aim: Limitations in available therapies for trypanosomiases indicate the need for improved medicines. Cysteine proteases cruzain and rhodesain are validated targets for treatment of Chagas disease and human African trypanosomiasis. Previous studies reported a benzimidazole series as potent cruzain inhibitors. Results & methodology: Considering the high similarity between these proteases, we evaluated 40 benzimidazoles against rhodesain. We describe their structure-activity relationships (SAR), revealing trends similar to those observed for cruzain and features that lead to enzyme selectivity. This series comprises noncovalent competitive inhibitors (best Ki = 0.21 μM against rhodesain) and micromolar activity against Trypanosoma brucei brucei. A cheminformatics analysis confirms scaffold novelty, and the inhibitors described have favorable predicted physicochemical properties. Conclusion: Our results support this series as a starting point for new human African trypanosomiasis medicines.

Schistosomiasis is a chronic and morbid disease of poverty affecting approximately 200 million people worldwide. Mature schistosome flatworms wander in the host's hepatic portal and mesenteric venous system where they encounter a range of blood flow conditions and geometrical confinement. However, the mechanisms that support schistosome locomotion and underlie the pathogen's adaptation to its physical environment are largely unknown. By combining microfabrication and traction force microscopy, we developed various in vitro assays to quantify the mechanics of locomotion of adult male Schistosoma mansoni in different physiologically relevant conditions. We show that in unconfined settings, the parasite undergoes two-anchor marching mediated by the coordinated action of its oral and ventral suckers. This mode of locomotion is maintained when the worm faces an external flow, to which it responds by adjusting the strength of its suckers. In geometrically confined conditions, S. mansoni switches to a different crawling modality by generating retrograde peristaltic waves along its body, a mechanism shared with terrestrial and marine worms. However, while the surface of most worms has backward-pointing bristles that rectify peristaltic waves and facilitate forward locomotion, S. mansoni has isotropically oriented tubercles. This requires tight coordination between muscle contraction and substrate friction but gives S. mansoni the ability to reverse its direction of locomotion without turning its body, which is likely advantageous to manoeuvre in narrow-bore vessels. We show that the parasite can also coordinate the action of its suckers with its peristaltic body contractions to increase crawling speed. Throughout this study, we report on a number of biomechanical parameters to quantify the motility of adult schistosomes (e.g. sucker grabbing strength, the rate of detachment under flow, peristaltic wave properties and traction stresses). The new series of in vitro assays make it possible to quantify key phenotypical aspects of S. mansoni motility that could guide the discovery of new drugs to treat schistosomiasis.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of administration. In an effort to develop new HAT therapeutics, we report the structure-activity relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We identified compound 18, which showed an improved kinase selectivity profile and acceptable pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease. We also report the results of cross-screening these compounds against T. cruzi, L. donovani, and S. mansoni.

Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs., Author summary Parasitic diseases are a major cause of human morbidity and mortality worldwide, as well as a significant economic drain in developing countries. Many parasites have limited treatment options with low efficacy and significant side effects, however research into new therapeutics suffers from a lack of investment. In this study, we characterize four potential anti-parasitic drugs: anisomycin, nithiamide, prodigiosin and obatoclax. These drugs were previously shown to effectively inhibit Entamoeba histolytica, the parasite that causes amebic dysentery. Here, we demonstrate that these drugs have activity against a wide variety of parasites from different taxonomic groups. Additionally, we assessed the speed of killing of these compounds against E. histolytica and the brain pathogen Balamuthia mandrillaris, and show that several are faster acting than current drugs. Two of these drugs (prodigiosin and obatoclax) had broad-spectrum activity, including against life stages not treated by current drugs such as juvenile schistosome worms, and three (obatoclax, nithiamide and anisomycin) have been used previously in humans. Although more study will be needed to adapt these drugs to the varying requirements for treatment of each parasitic disease, this work is a promising beginning towards identifying drugs against multiple parasites that are human pathogens.

In vitro whole-organism screens of Trypanosoma brucei with representative examples of brain-penetrant microtubule (MT)-stabilizing agents identified lethal triazolopyrimidines and phenylpyrimidines with sub-micromolar potency. In mammalian cells, these antiproliferative compounds disrupt MT integrity and decrease total tubulin levels. Their parasiticidal potency, combined with their generally favorable pharmacokinetic properties, which include oral bioavailability and brain penetration, suggest that these compounds are potential leads against human African trypanosomiasis.

Neglected tropical diseases caused by metazoan parasites are major public health concerns, and therefore, new methods for their control and elimination are needed. Research over the last 25 years has revealed the vital contribution of cysteine proteases to invasion of and migration by (larval) helminth parasites through host tissues, in addition to their roles in embryogenesis, molting, egg hatching, and yolk degradation. Their central function to maintaining parasite survival in the host has made them prime intervention targets for novel drugs and vaccines. This review focuses on those helminth cysteine proteases that have been functionally characterized during the varied early stages of development in the human host and embryogenesis.

We briefly review cysteine proteases (orthologs of mammalian cathepsins B, L, F, and C) that are expressed in flatworm and nematode parasites. Emphasis is placed on enzyme activities that have been functionally characterized, are associated with the parasite gut, and putatively contribute to degrading host proteins to absorbable nutrients [1–4]. Often, gut proteases are expressed as multigene families, as is the case with Fasciola [5] and Haemonchus [6], presumably expanding the range of substrates that can be degraded, not least during parasite migration through host tissues [5]. The application of the free-living planarian and Caenorhabditis elegans as investigative models for parasite cysteine proteases is discussed. Finally, because of their central nutritive contribution, targeting the component gut proteases with small-molecule chemical inhibitors and understanding their utility as vaccine candidates are active areas of research [7].

In the search for new anti-schistosomal agents, a series of fifteen ortho-nitrobenzyl derivatives was assayed in vitro against both the schistosomulum (somule) and adult forms of Schistosoma mansoni. Compounds 8 and 12 showed significant activity against somules at low micromolar concentrations, but none was active against adults. The SAR demonstrated that the compounds most active against the parasite were mutagenic to the human cell line RKO-AS45-1 only at concentrations 10- to 40-fold higher than the worm-killing dose. Given their electrophilicity, compounds were also screened as inhibitors of the S. mansoni cysteine protease (cathepsin B1) in vitro. Amides 5 and 15 exhibited a modest inhibition activity with values of 55.7 and 50.6 % at 100 µM, respectively. The nitrobenzyl compounds evaluated in this work can be regarded as hits in the search for more active and safe anti-schistosomal agents.

Schistosomiasis is a tropical disease caused by a flatworm trematode parasite that infects over 200 million people worldwide. Treatment and control of the disease rely on just one drug, praziquantel. The possibility of drug resistance coupled with praziquantel's variable efficacy encourages the identification of new drugs and drug targets. Disruption of neuromuscular homeostasis in parasitic worms is a validated strategy for drug development. In schistosomes, however, much remains to be understood about the organization of the nervous system, its component neurotransmitters and potential for drug discovery. Using synapsin as a neuronal marker, we map the central and peripheral nervous systems in the Schistosoma mansoni adult and schistosomulum (post-infective larva). We discover the widespread presence of octopamine (OA), a tyrosine-derived and invertebrate-specific neurotransmitter involved in neuromuscular coordination. OA labeling facilitated the discovery of two pairs of ganglia in the brain of the adult schistosome, rather than the one pair thus far reported for this and other trematodes. In quantitative phenotypic assays, OA and the structurally related tyrosine-derived phenolamine and catecholamine neurotransmitters differentially modulated schistosomulum motility and length. Similarly, from a screen of 28 drug agonists and antagonists of tyrosine-derivative signaling, certain drugs that act on OA and dopamine receptors induced robust and sometimes complex concentration-dependent effects on schistosome motility and length; in some cases, these effects occurred at concentrations achievable in vivo. The present data advance our knowledge of the organization of the nervous system in this globally important pathogen and identify a number of drugs that interfere with tyrosine-derivative signaling, one or more of which might provide the basis for a new chemotherapeutic approach to treat schistosomiasis. This article has an associated First Person interview with the first author of the paper.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

The last half-century has provided all of the (few) drugs currently used to treat human helminthiases. Concern regarding the long-term utility of these drugs, given how readily resistance evolves in the veterinary-agricultural sector, spurs the discovery of new chemical entities. We review the approaches and technologies in use to identify anthelmintics and discuss a number of drug discovery paradigms that may prove pivotal to the next half-century of anthelmintic development.

Background Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer’s disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). Methodology/Principal findings A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite’s genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target. Conclusions/Significance The orthogonal chemical, biological and genetic strategies employed identify SmPDE4A’s contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets., Author summary Just one drug, praziquantel (PZQ), is available to treat schistosomiasis, a flatworm disease that infects over 240 million people, mainly in Africa. With the expanding distribution of PZQ, and the associated threat of drug resistance, new drugs and drug targets are needed. We screened Schistosoma mansoni worms with over 1,000 benzoxaborole chemical molecules from Anacor Pharmaceuticals to identify a subset of human cyclic nucleotide phosphodiesterase 4 (huPDE4) inhibitors that cause parasite hypermotility and degeneration. We identified four PDE4 genes in the genome of the parasite and recombinantly expressed one of them (SmPDE4A) in bacteria. This enzyme was then used to uncover a relationship between the degree of enzyme inhibition, and the generation of parasite hypermotility and degeneration. To understand whether the SmPDE4A enzyme is the target of the benzoxaboroles in the parasite, we incorporated the coding DNA for SmPDE4A into the model nematode Caenorhabditis elegans that lacked its own functional PDE4 and, as a consequence, was hypermotile. These ‘transgenic’ worms displayed normal motility which could be increased by applying the most potent huPDE4 benzoxaborole inhibitors. In summary, the chemical, biological and genetic approaches taken identify SmPDE4A as a potential drug target for treating schistosomiasis. The potential value of C. elegans as a tool to test and optimize therapeutic chemistries for a flatworm disease is also highlighted.

Human hookworm Necator americanus causes iron deficiency anemia, as the parasite ingests blood from the gastrointestinal tract of its human host. The virulence factors of this blood feeding nematode have not been researched extensively. This bioinformatics based study focuses on eight of the cathepsin B like cysteine proteases (CPs) of the worm, which could have immense pathogenic potential. The role of the individual CPs remain vaguely determined except for CP3 which has been shown to act as globinase in the hemoglobin degradation pathway. In this study, the cysteine proteases were subjected to predictive molecular characterizations viz: probability of extracellular secretion to the interface between pathogen and host, ability for hemoglobin degradation, and/or interaction with host plasma proteins. CP1-CP6, which harbored the active site cysteine and were observed to have N terminal signal peptide for extracellular localization, were relevantly predicted to be secretory. Amongst these, CP2 and CP3 showed the presence of hemoglobinase motif derived in this study that could be a prerequisite for globin or hemoglobin degradation. CP1 and CP6 that shared similar S2 subsite features with kininogen-cleaving cathepsin B and cruzain, suggests the possible involvement of these two hookworm CPs in binding and cleaving High Molecular weight kininogen (HMWK). CP1, CP2, CP3, CP5 and CP6 were predicted to bind heparin, which is the glycosaminoglycan molecule that has been demonstrated to aid the substrate-cleaving functionality of other cysteine proteases like human cathepsin B and cruzain. Heparin docked onto the NA CPs at the C terminal domain, away from the active site, similar to what has been shown for heparin binding to cathepsin B and cruzain. These observations therefore lead us to the hypothesize that the functions of the hookworm CPs, which could include preventing blood clot formation, might be assisted by heparin. Also, this study underscores the potential of synthetic heparin analogs as molecular treatment for hookworm infection, which could have implications for future drug design.

Blood-protein digestion is a key physiological process providing essential nutrients for ticks and is a prerequisite for the transmission of tick-borne pathogens. Recently, substantial progress has been made in determining the proteolytic machinery in tick gut tissue, which is based on a dynamic multienzyme network capable of processing a vast amount of host blood. In this article we summarize our current knowledge of the molecular mechanisms of tick hematophagy and their similarities to those of Platyhelminthes, nematodes, and Plasmodium. Future research perspectives, including the potential for rational control of ticks and transmitted diseases, are also discussed.

Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1′ position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1′ regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2′. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.