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.

Parasitic flatworms express a diverse array of G protein coupled receptors, but our knowledge of their pharmacological profile is limited. No high throughput screen of a flatworm GPCR has been reported, even though these targets have precedent for high druggability and functionality in the chemotherapeutically vulnerable excitable cell niche. The goal of this study was to establish a method for profiling flatworm G protein coupled receptors that can be scaled to high content screening. Using a cAMP biosensor, we have performed a proof of principle miniaturized screen on a schistosome serotonergic GPCR that resolves new ligands that potently and selectivity block 5-HT receptor activity in vitro, and 5-HT evoked responses in schistosomules and adult worms. This approach evidences the pharmacological divergence of a parasitic GPCR from the closest human homolog and a capacity for high content interrogation of flatworm GPCR properties and ligand specificities.

Funding: Work in the Marchant lab was supported by the NIH (GM088790) and a University of Minnesota Medical School Innovation Research Grant. Work in the Roth Lab was supported by NIH (PDSP and R01MH61887 to JDM and BLR). JDC was supported by Stem Cell Biology Training Grant (T32GM113846). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2016 Chan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To evaluate this technology, we applied this approach to pharmacologically profile a S. mansoni serotonergic GPCR (Sm.5HTR) that has been shown in vitro to respond to 5-HT through elevation of cAMP [ 20 ]. Sm.5HTR is the parasitic homologue of the planarian serotonergic GPCR (S7.1) that we have recently shown modulates the efficacy of PZQ in vivo [ 13 ]. However, as with most flatworm GPCRs, little is known about the pharmacology of this receptor. An initial characterization revealed blockade of 5-HT evoked signals in the presence of high concentrations (100μM) of mammalian bioaminergic blockers [ 20 ]. Here, we have applied the GloSensor assay in a proof of principle pilot screen for flatworm GPCR modulation. Our data evidence the extent of pharmacological divergence between the schistosome receptor and the human 5-HT 7 -receptor homolog (Hs.5HT7R), and reveal new ligands and compound series selective for the parasitic GPCR. Finally, despite these differences in ligand selectivity, we demonstrate conservation of an unusual antagonist-evoked inactivation mechanism for Sm.5HTR, a pharmacological phenomenon also exhibited at Hs.5HT7R [ 25 , 26 ], where exposure to a subset of antagonists results in a prolonged inactivation of signaling activity from the receptor. This property enhances the attractiveness of Sm.5HTR as an anthelmintic drug target.

Therefore the goal of this study was to establish a method for profiling flatworm GPCRs that can be effectively scaled to HTS. Our priorities for a platform were: first, a robustness for miniaturization into a multiwall plate format to permit chemical library screening, and second, use of a proximal readout of receptor activity within intact cells to enable real time monitoring of GPCR activity that can resolve different types of modulators (full, partial and inverse agonists, allosteric modulators). One technology that fulfills these requirements employs a bioluminescent cAMP reporter to monitor the activity of G s and G i -coupled GPCRs, marketed as GloSensor. The assay is based upon a crucially permutated form of firefly luciferase incorporating a cAMP-binding domain from PKA, such that cAMP-binding causes a conformational change in the enzyme that enhances the luminescent signal [ 23 ]. The dynamic range and sensitivity of the biosensor has been shown to be compatible with a variety of HTS assays [ 23 , 24 ].

This is an important realization as flatworm G protein coupled receptors (GPCRs) are logical candidates for antischistosomal drug development efforts. Over one quarter of current therapeutics target rhodopsin-like GPCRs [ 16 ]. However, barriers have been a lack of understanding of the physiology of specific GPCRs from within the broad GPCR portfolio (~75–120 in S. mansoni [ 17 – 19 ]) expressed by these organisms, as well as struggles optimizing functional expression of individual flatworm GPCRs in heterologous assay systems. However several groups have now begun to define a role for specific GPCRs within the chemotherapeutically vulnerable excitable cell niche [ 13 , 20 – 22 ], highlighting the key challenge of optimizing robust platforms for pharmacologically profiling these GPCRs in a miniaturized format compatible with high throughput screening (HTS). To our knowledge, no library screen of a flatworm GPCR has yet been reported. Prior studies have simply relied on interrogation of expressed GPCRs against handfuls of ligands selected around inferred agonist specificity.

A logical place to start is with downstream effectors within the broader PZQ interactome. Over the last few years, therefore, our laboratory has attempted to bring fresh perspective to understand how PZQ works based upon a serendipitous basic science finding. During regeneration of the planarian flatworm D. japonica–a widely used regenerative biology model [ 10 ]–PZQ miscued polarity signaling to cause regeneration of bipolar (‘two-headed’) worms with dual, integrated organ systems [ 11 ]. This visually striking phenotype, coupled with the tractability of the planarian system to in vivo RNAi, allowed us to progressively define pathways engaged by PZQ in vivo [ 11 – 14 ]. These studies culminated in a model where PZQ acts as an ergomimetic [ 13 ] with in vivo PZQ efficacy regulated by the opposing functionality of dopaminergic and serotonergic neurons [ 11 – 14 ], known regulators of muscular activity, the tissue where planarian polarity determinants reside [ 15 ]. The serotonergic and dopaminergic G protein coupled receptors (GPCRs) engaged by activity of these bioaminergic neurons therefore represent potential downstream PZQ effectors. Their engagement by ligands, as shown for bromocriptine and other ergot alkaloids, phenocopy PZQ action in vivo [ 13 , 14 ].

PZQ was originally developed during the 1970s, and the continued effectiveness of this agent over four decades of usage for treating a variety of parasitic infections has proven critically impactful [ 1 ]. Indeed this clinical efficacy has ironically proven to be a factor that has restrained efforts to develop alternative therapies, and at the most basic level, define how PZQ works. However several features of PZQ remain less than ideal and require improvement. First, our lack of mechanistic understanding of how PZQ works has proved a roadblock in the rational design of new drugs. There is a need to identify new druggable targets that exploit broader vulnerabilities within PZQ-sensitive pathways [ 2 – 4 ]. Second, our inability to improve on PZQ by chemical derivatization of the drug: all PZQ derivatives synthesized to date are less effective than the parent compound. The need is to identify novel structural pharmacophores that impair parasite viability. Third, the inability of PZQ to kill all parasitic life cycle stages. Juvenile worms are refractory to PZQ [ 5 , 6 ], possibly a contributory factor driving development of drug resistance [ 5 , 7 ]. The need is to identify new targets expressed throughout all lifecycle stages that are ideally conserved in other PZQ-sensitive parasites. Fourth, sub-optimal cure rates in the field: PZQ requires multiple drug dosings to achieve maximal cure rates for schistosomiasis, a regimen which is not always executed in mass drug administration efforts [ 8 , 9 ]. Therefore, there is clear opportunity to improve on the clinical penetrance of PZQ. These issues support efforts to identify new, druggable targets for development of next generation anthelmintics.

The neglected tropical disease Schistosomiasis is the most socioeconomically devastating helminth infection, and the second most burdensome parasitic infection behind malaria, infecting over 200 million people worldwide [ 1 ]. Infected individuals are treated by the drug praziquantel (PZQ), the mainstay therapeutic for disease control.

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