Development of non-sedating benzodiazepines with in vivo antischistosomal activity.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad-spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970s. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) is not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Phenotypic Profiling of Macrocyclic Lactones on Parasitic Schistosoma Flatworms.

Macrocyclic lactones are front-line therapies for parasitic roundworm infections; however, there are no comprehensive studies on the activity of this drug class against parasitic flatworms. Ivermectin is well known to be inactive against flatworms. However, the structure-activity relationship of macrocyclic lactones may vary across phyla, and it is entirely possible other members of this drug class do in fact show antiparasitic activity on flatworms. For example, there are several reports hinting at the anti-schistosomal activity of doramectin and moxidectin. To explore this class further, we developed an automated imaging assay combined with measurement of lactate levels from worm media. This assay was applied to the screening of 21 macrocyclic lactones (avermectins, milbemycins, and others such as spinosyns) against adult schistosomes. These in vitro assays identified several macrocyclic lactones (emamectin, milbemycin oxime, and the moxidectin metabolite 23-ketonemadectin) that caused contractile paralysis and lack of lactate production. Several of these were also active against miracidia, which infect the snail intermediate host. Hits prioritized from these in vitro assays were administered to mice harboring patent schistosome infections. However, no reduction in worm burden was observed. Nevertheless, these data show the utility of a multiplexed in vitro screening platform to quantitatively assess drug action and exclude inactive compounds from a chemical series before proceeding to in vivo studies. While the prototypical macrocyclic lactone ivermectin displays minimal activity against adult Schistosoma mansoni, this family of compounds does contain schistocidal compounds which may serve as a starting point for development of new anti-flatworm chemotherapies.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca2+ influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use. Here, we demonstrate that PZQ activates a schistosome transient receptor potential (TRP) channel, christened SmTRPMPZQ, present in parasitic schistosomes and other PZQ-sensitive parasites. Several properties of SmTRPMPZQ were consistent with known effects of PZQ on schistosomes, including (i) nanomolar sensitivity to PZQ; (ii) stereoselectivity toward (R)-PZQ; (iii) mediation of sustained Ca2+ signals in response to PZQ; and (iv) a pharmacological profile that mirrors the well-known effects of PZQ on muscle contraction and tegumental disruption. We anticipate that these findings will spur development of novel therapeutic interventions to manage schistosome infections and broader interest in PZQ, which is finally unmasked as a potent flatworm TRP channel activator.

Utilizing the planarian voltage-gated ion channel transcriptome to resolve a role for a Ca2+ channel in neuromuscular function and regeneration.

The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca2+ signaling in planarian regeneration. Here, to facilitate study of acute signaling events in the excitable cell niche, we provide a de novo transcriptome assembly from the planarian Dugesia japonica allowing characterization of the diverse ionotropic portfolio of this model organism. We demonstrate the utility of this resource by proceeding to characterize the individual role of each of the planarian voltage-operated Ca2+ channels during regeneration, and demonstrate that knockdown of a specific voltage operated Ca2+ channel (Cav1B) that impairs muscle function uniquely creates an environment permissive for anteriorization. Provision of the full transcriptomic dataset should facilitate further investigations of molecules within the planarian voltage-gated channel portfolio to explore the role of excitable cell physiology on regenerative outcomes. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors.

Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets ('target selection') and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics.

'Death and axes': unexpected Ca²âº entry phenologs predict new anti-schistosomal agents.

Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²âº channel modulates opposing dopaminergic and serotonergic pathways to regulate 'head' structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²âº entry mechanism, and comprise unexpected Ca²âº phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents.

Long Non-Coding RNA Levels Are Modulated in Schistosoma mansoni following In Vivo Praziquantel Exposure.

Schistosomiasis is a disease caused by trematodes of the genus Schistosoma that affects over 200 million people worldwide. For decades, praziquantel (PZQ) has been the only available drug to treat the disease. Despite recent discoveries that identified a transient receptor ion channel as the target of PZQ, schistosome response to this drug remains incompletely understood, since effectiveness relies on other factors that may trigger a complex regulation of parasite gene expression. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that play important roles in S. mansoni homeostasis, reproduction, and fertility. Here, we show that in vivo PZQ treatment modulates lncRNA levels in S. mansoni. We re-analyzed public RNA-Seq data from mature and immature S. mansoni worms treated in vivo with PZQ and detected hundreds of lncRNAs differentially expressed following drug exposure, many of which are shared among mature and immature worms. Through RT-qPCR, seven out of ten selected lncRNAs were validated as differentially expressed; interestingly, we show that these lncRNAs are not adult worm stage-specific and are co-expressed with PZQ-modulated protein-coding genes. By demonstrating that parasite lncRNA expression levels alter in response to PZQ, this study unravels an important step toward elucidating the complex mechanisms of S. mansoni response to PZQ.

Development of non-sedating antischistosomal benzodiazepines.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970's. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Schistosomes contain divergent ligand-gated ion channels with an atypical Cys-loop motif.

Ligand-gated ion channels (LGICs) are important regulators of neuromuscular function, making them attractive antiparasitic drug targets. While roundworm LGICs are targeted by several anthelmintic classes, flatworm LGICs are less studied. Chromosome-level genome assemblies have recently been released for Schistosoma flatworm species that cause the disease schistosomiasis. These have allowed us to comprehensively predict schistosome LGICs, adding to prior annotations. Analysis of LGIC sequences revealed a clade of receptors lacking cysteines at the eponymous Cys-loop region of the channel. Since these atypical channels are divergent from mammalian LGICs, they may be promising targets to treat diseases caused by parasitic flatworms.

Annotation of G-Protein Coupled Receptors in the Genomes of Parasitic Blood Flukes.

Infection with Schistosoma parasitic flatworms ( Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum ) causes the neglected tropical disease schistosomiasis. There is a need to identify new chemotherapies to treat these parasites, and G-protein coupled receptors (GPCRs) are a logical druggable targets to explore given they control key aspects of schistosome biology such as neuromuscular function and reproduction. Updated chromosome level genome assemblies for each of the three major species have recently been released. However, studies on these GPCRs require accurate, updated genome annotations. Here, we have re-annotated the GPCRs present in each of the three major schistosome species.

Multivariate chemogenomic screening prioritizes new macrofilaricidal leads.

Development of direct acting macrofilaricides for the treatment of human filariases is hampered by limitations in screening throughput imposed by the parasite life cycle. In vitro adult screens typically assess single phenotypes without prior enrichment for chemicals with antifilarial potential. We developed a multivariate screen that identified dozens of compounds with submicromolar macrofilaricidal activity, achieving a hit rate of >50% by leveraging abundantly accessible microfilariae. Adult assays were multiplexed to thoroughly characterize compound activity across relevant parasite fitness traits, including neuromuscular control, fecundity, metabolism, and viability. Seventeen compounds from a diverse chemogenomic library elicited strong effects on at least one adult trait, with differential potency against microfilariae and adults. Our screen identified five compounds with high potency against adults but low potency or slow-acting microfilaricidal effects, at least one of which acts through a novel mechanism. We show that the use of microfilariae in a primary screen outperforms model nematode developmental assays and virtual screening of protein structures inferred with deep learning. These data provide new leads for drug development, and the high-content and multiplex assays set a new foundation for antifilarial discovery.

Opposing roles of voltage-gated Ca2+ channels in neuronal control of regenerative patterning.

There is intense interest in developing methods to regulate proliferation and differentiation of stem cells into neuronal fates for the purposes of regenerative medicine. One way to do this is through in vivo pharmacological engineering using small molecules. However, a key challenge is identification of relevant signaling pathways and therein druggable targets to manipulate stem cell behavior efficiently in vivo. Here, we use the planarian flatworm as a simple chemical-genetic screening model for nervous system regeneration to show that the isoquinoline drug praziquantel (PZQ) acts as a small molecule neurogenic to produce two-headed animals with integrated CNSs following regeneration. Characterization of the entire family of planarian voltage-operated Ca(2+) channel α subunits (Ca(v)α), followed by in vivo RNAi of specific Ca(v) subunits, revealed that PZQ subverted regeneration by activation of a specific voltage-gated Ca(2+) channel isoform (Ca(v)1A). PZQ-evoked Ca(2+) entry via Ca(v)1A served to inhibit neuronally derived Hedgehog signals, as evidenced by data showing that RNAi of Ca(v)1A prevented PZQ-evoked bipolarity, Ca(2+) entry, and decreases in wnt1 and wnt11-5 levels. Surprisingly, the action of PZQ was opposed by Ca(2+) influx through a closely related neuronal Ca(v) isoform (Ca(v)1B), establishing a novel interplay between specific Ca(v)1 channel isoforms, Ca(2+) entry, and neuronal Hedgehog signaling. These data map PZQ efficacy to specific neuronal Ca(v) complexes in vivo and underscore that both activators (Ca(v)1A) and inhibitors (Ca(v)1B) of Ca(2+) influx can act as small molecule neurogenics in vivo on account of the unique coupling of Ca(2+) channels to neuronally derived polarity cues.

wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms.

Advances in high-throughput and high-content imaging technologies require concomitant development of analytical software capable of handling large datasets and generating relevant phenotypic measurements. Several tools have been developed to analyze drug response phenotypes in parasitic and free-living worms, but these are siloed and often limited to specific instrumentation, worm species, and single phenotypes. No unified tool exists to analyze diverse high-content phenotypic imaging data of worms and provide a platform for future extensibility. We have developed wrmXpress, a unified framework for analyzing a variety of phenotypes matched to high-content experimental assays of free-living and parasitic nematodes and flatworms. We demonstrate its utility for analyzing a suite of phenotypes, including motility, development/size, fecundity, and feeding, and establish the package as a platform upon which to build future custom phenotypic modules. We show that wrmXpress can serve as an analytical workhorse for anthelmintic screening efforts across schistosomes, filarial nematodes, and free-living model nematodes and holds promise for enabling collaboration among investigators with diverse interests.

High-content approaches to anthelmintic drug screening.

Most anthelmintics were discovered through in vivo screens using animal models of infection. Developing in vitro assays for parasitic worms presents several challenges. The lack of in vitro life cycle culture protocols requires harvesting worms from vertebrate hosts or vectors, limiting assay throughput. Once worms are removed from the host environment, established anthelmintics often show no obvious phenotype - raising concerns about the predictive value of many in vitro assays. However, with recent progress in understanding how anthelmintics subvert host-parasite interactions, and breakthroughs in high-content imaging and machine learning, in vitro assays have the potential to discern subtle cryptic parasite phenotypes. These may prove better endpoints than conventional in vitro viability assays.

Schistosoma mansoni alter transcription of immunomodulatory gene products following in vivo praziquantel exposure.

Control of the neglected tropical disease schistosomiasis relies almost entirely on praziquantel (PZQ) monotherapy. How PZQ clears parasite infections remains poorly understood. Many studies have examined the effects of PZQ on worms cultured in vitro, observing outcomes such as muscle contraction. However, conditions worms are exposed to in vivo may vary considerably from in vitro experiments given the short half-life of PZQ and the importance of host immune system engagement for drug efficacy in animal models. Here, we investigated the effects of in vivo PZQ exposure on Schistosoma mansoni. Measurement of pro-apoptotic caspase activation revealed that worm death occurs only after parasites shift from the mesenteric vasculature to the liver, peaking 24 hours after drug treatment. This indicates that PZQ is not directly schistocidal, since PZQ's half-life is ~2 hours in humans and ~30 minutes in mice, and focuses attention on parasite interactions with the host immune system following the shift of worms to the liver. RNA-Seq of worms harvested from mouse livers following sub-lethal PZQ treatment revealed drug-evoked changes in the expression of putative immunomodulatory and anticoagulant gene products. Several of these gene products localized to the schistosome esophagus and may be secreted into the host circulation. These include several Kunitz-type protease inhibitors, which are also found in the secretomes of other blood feeding animals. These transcriptional changes may reflect mechanisms of parasite immune-evasion in response to chemotherapy, given the role of complement-mediated attack and the host innate/humoral immune response in parasite elimination. One of these isoforms, SmKI-1, has been shown to exhibit immunomodulatory and anti-coagulant properties. These data provide insight into the effect of in vivo PZQ exposure on S. mansoni, and the transcriptional response of parasites to the stress of chemotherapy.

An efficient debromination technique using PMHS with a number of ligands containing different functional groups.

Herein is described the strategy to debrominate different aryl bromides selectively, using polymethylhydrosiloxane (PMHS) which tolerates a variety of functional groups. Key elements of this approach include the use of catalytic Pd(OAc)2 and the correct equivalents of polymethylhydrosiloxane (PMHS), in conjunction with aqueous KF. The present reaction process provides a strategic tool for the synthesis of a number of medicinally important molecules.

Anti-schistosomal action of the calcium channel agonist FPL-64176.

Subversion of parasite neuromuscular function is a key strategy for anthelmintic drug development. Schistosome Ca2+ signaling has been an area of particular interest for decades, with a specific focus on L-type voltage-gated Ca2+ channels (Cavs). However, the study of these channels has been technically challenging. One barrier is the lack of pharmacological probes that are active on flatworms, since the dihydropyridine (DHP) based ligands typically used to study Cavs are relatively ineffective on schistosomes. Here, we have characterized the effect of a structurally distinct putative L-type Cav agonist, FPL-64176, on schistosomes cultured ex vivo and in an in vivo murine model of infection. Unlike DHPs, FPL-64176 evokes rapid and sustained contractile paralysis of adult Schistosoma mansoni reminiscent of the anthelmintic praziquantel. This is accompanied by tegument disruption and an arrest of mitotic activity in somatic stem cells and germ line tissues. Interestingly, this strong ex vivo phenotype was temperature dependent, with FPL-64176 treatment being less potent at 37 °C than 23 °C. However, FPL-64176 caused intra-tegument lesions at the basement membrane of worms cultured ex vivo under both conditions, as well as an in vivo hepatic shift of parasites from the mesenteric vasculature of infected mice to the liver. Gene expression profiling of worms harvested following in vivo FPL-64176 exposure reveals differences in transcripts associated with muscle and extracellular matrix function, as well as female reproduction, which is consistent with the worm phenotypes observed following ex vivo drug treatment. These data advance FPL-64176 as a useful tool to study schistosome Ca2+ signaling, and the benzoyl pyrrole core as a hit compound that may be optimized to develop new parasite-selective leads.

Non-sedating benzodiazepines cause paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni.

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports on cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines given the anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970's but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZ's anti-parasitic target is distinct from the human receptors that drive sedation. Therefore, we have screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, resulting in extensive vacuole formation beneath the apical membrane. The hit compound series identified has a dramatically lower (~1000×) affinity for the human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects.

Coalescing beneficial host and deleterious antiparasitic actions as an antischistosomal strategy.

Conventional approaches for antiparasitic drug discovery center upon discovering selective agents that adversely impact parasites with minimal host side effects. Here, we show that agents with a broad polypharmacology, often considered 'dirtier' drugs, can have unique efficacy if they combine deleterious effects on the parasite with beneficial actions in the host. This principle is evidenced through a screen for drugs to treat schistosomiasis, a parasitic flatworm disease that impacts over 230 million people. A target-based screen of a Schistosoma serotoninergic G protein coupled receptor yielded the potent agonist, ergotamine, which disrupted worm movement. In vivo, ergotamine decreased mortality, parasite load and intestinal egg counts but also uniquely reduced organ pathology through engagement of host GPCRs that repressed hepatic stellate cell activation, inflammatory damage and fibrosis. The unique ability of ergotamine to engage both host and parasite GPCRs evidences a future strategy for anthelmintic drug design that coalesces deleterious antiparasitic activity with beneficial host effects.

Structure-activity profiling of alkaloid natural product pharmacophores against a Schistosoma serotonin receptor.

Serotonin (5-HT) is an important regulator of numerous aspects of flatworm biology, ranging from neuromuscular function to sexual maturation and egg laying. In the parasitic blood fluke Schistosoma mansoni, 5-HT targets several G-protein coupled receptors (GPCRs), one of which has been demonstrated to couple to cAMP and regulate parasite movement. This receptor, Sm.5HTRL, has been successfully co-expressed in mammalian cells alongside a luminescent cAMP-biosensor, enabling pharmacological profiling for candidate anti-schistosomal drugs. Here, we have utilized this assay to perform structure-activity investigations of 143 compounds containing previously identified alkaloid natural product pharmacophores (tryptamines, aporphines and protoberberines) shown to regulate Sm.5HTRL. These experiments mapped regions of the tryptamine pharmacophore amenable and intolerant to substitution, highlighting differences relative to orthologous mammalian 5-HT receptors. Potent Sm.5HTRL antagonists were identified, and the efficacy of these compounds were evaluated against live adult parasites cultured ex vivo. Such structure-activity profiling, characterizing the effect of various modifications to these core ring systems on Sm.5HTRL responses, provides greater understanding of pharmacophores selective for this target to aid future drug development efforts.