Msc Chemistry Molecular Sciences Literature Thesis Functional and Molecular Characterization of G Protein-Coupled Receptors In

MSc Chemistry Molecular Sciences

Literature Thesis

Functional and molecular characterization of G protein-coupled receptors in Schistosoma mansoni

Exploring the possibility of schistosome GPCRs as anthelmintic targets

Roxane Biersteker 10808272 (UvA) / 2565601 (VU)

July 2020 12 EC

Supervisor/Examiner: Department Research Institute: Prof. dr. R. Leurs Medicinal chemistry (AIMMS) dr. H.F. Vischer Abstract Schistosomiasis is one of the most devastating tropical diseases, affecting at least 230 million people. Currently, no vaccine exists to treat this disease and the only drug available is praziquantel. This drug is inactive against juvenile schistosomes and the first signs of resistance of S. mansoni to PZQ have been observed. Therefore, there is an urgent need to identify new drug targets. This paper has explored the possibility of schistosome GPCRs as anthelmintic targets. This paper showed that all major GPCR subfamilies and a flatworm specific GPCR family (PROF) are represented in the genome of S. mansoni. Analysis of GPCR transcription profiles revealed that GPCRs are involved in non-gonad, pairing-dependent processes as well as in gonad-specific functions. Furthermore, this paper has shown that schistosome GPCRs are expressed in various life-stages, and several receptors are upregulated in schistosomula. This paper has analyzed the characterization of all deorphanized GPCRs in S. mansoni. These included histamine, dopamine, acetylcholine, glutamate and serotonin receptors. Immunolocalization and RNAi studies suggest that these receptors play a role in worm motility and/or oogenesis. The majority of the deorphanized receptors have different pharmacological profiles than those of receptors in the mammalian host. This indicates that schistosome GPCRs have great potential for parasite-selective drug targeting. The first flatworm library screen of a flatworm GPCR has been described and indicated that structure-activity profiling can provide a deeper understanding of pharmacophores that are selective for parasite receptors. Schistosome GPCRs are promising antischistosomal drug targets because they are expressed in various life-stages, both sexes and various tissues, including the gonads. Therefore, compounds that target these receptors may be used to combat the acute as well as the chronic phase of schistosomiasis, and prevent the dissemination of schistosome eggs.

Index

Introduction 4

1. Schistosoma mansoni 6 1.1 Lifecycle and pathogenesis 6 1.2 Nervous system of S. mansoni 8 1.3 Summary 9

2. GPCR (sub)families in S. mansoni 10 2.1 All major GPCR families are present in S. mansoni 12 2.2 Rhodopsin family GPCRs 14 2.3 Platyhelminth-specific Rhodopsin-like orphan family 15 2.4 Adhesion/Secretin family GPCRs 18 2.5 Glutamate family GPCRs 18 2.6 Frizzled family GPCRs 20 2.7 Summary 20

3. Tissue-specific and pairing dependent GPCR expression in S. mansoni 22 3.1 Analysis of tissue-specific GPCR expression in paired and unpaired schistosomes 22 3.2 S. mansoni GPCRs contribute to non-gonad, pairing-dependent processes 23 3.3 S. mansoni GPCRs play a role in gonad-specific functions 25 3.4 Summary 27

4. GPCR expression in various S. mansoni life-cycle stages 28 4.1 GPCRs are low-abundantly expressed in adult schistosomes 28 4.2 Biogenic amine receptors are upregulated in parasitic stages 30 4.3 The larval nervous system undergoes broad changes during its development 32 4.4 Increased expression of PROF complements during juvenile life stages 33 4.5 Summary 35

5. Deorphanized S. mansoni GPCRs 36 5.1 Glutamate receptor SmGluR 39 5.2 Acetylcholine receptor SmGAR 42 5.3 Dopamine receptor SmD2 48 5.4 SmGPR-like receptors 52 5.5 Serotonin receptor Sm5HTR 75 5.6 Summary 80

6. Pharmacology of GPCRs in S. mansoni 81 6.1 Pharmacological profile of SmGluR 81 6.2 Pharmacological profile of SmGAR 83 6.3 Pharmacological profile of SmD2 85 6.4 Drug profiles of SmGPR-like receptors 87 6.5 Pharmacological profile of Sm5HTR 92 6.6 Repurpose promethazine as an antischistosomal drug 99 6.7 Summary 106

7. Discussion 108

8. References 124

Introduction

Schistosomiasis afflicts at least 230 million people in the (sub)tropics and is caused by flatworms of the genus Schistosoma.1 Acute schistosomiasis is characterized by abdominal pain, fatigue, fever, malaise and myalgia.2 Active and late chronic disease occurs because of immunopathological reactions against eggs that are retained in host tissue. These reactions result in an inflammatory and obstructive disease. The flatworm causes systemic morbidities that are associated with continuous inflammation due to multiple infections during the child’s early life. These include cognitive impairment, decreased aerobic capacity, anemia and growth stunting.1,3–6 Symptoms often persist after the infection has been terminated, especially in humans that have been infected in early life.1,7

Currently, no vaccine exists and the only drug available to cure this neglected parasitic disease is praziquantel (PZQ), which displays various disadvantages. For example, it is inactive against juvenile schistosomes and it needs to be administered multiple times in order to reach optimal cure rates.8,9,10 Chemical derivatives of PZQ all proved to be less effective and the lack of understanding of the mechanism of action hampers improvements in the efficacy.11 Moreover, the treatment of schistosomiasis will become increasingly difficult due to the emerging resistance of PZQ.12 For these reasons, there is an urgent need to identify new drug targets that are expressed throughout all life-stages of Schistosoma mansoni (S. mansoni).

Since G-protein coupled receptors (GPCRs) are the targets of 30–40% of currently marketed drugs for humans, they are an obvious target to explore for developing new anthelmintics.13,14 However, very little is known about platyhelminth GPCRs. In silico analyses showed that all major GPCR subfamilies are present in S. mansoni, including a rhodopsin subfamily specific to Platyhelminthes.15,16 Although only a limited number of schistosome GPCRs have been functionally characterized, the variety of GPCR genes suggests their involvement in a wide array of functions.17 GPCRs that play a role in reproduction are interesting potential drug targets because schistosome egg production is crucial for life cycle completion as well as for inducing the morbidity caused by schistosome infections.18,19 However, a deeper understanding of the roles of GPCR signaling in schistosome biology is necessary in order to identify candidate receptors.

The aim of this paper is to explore the possibility of schistosome GPCRs as anthelmintic targets. This study investigates the expression of schistosome GPCRs in various S. mansoni life-stages and tissues, and analyzes their functional characterization. Furthermore, this paper assesses the druggability potential of schistosome GPCRs by analyzing their pharmacological profiles and comparing them to those of mammalian GPCRs. In addition, this paper traces the development of a miniaturized screening assay and potential parasite-selective inhibitors.

This paper begins by discussing the schistosome lifecycle and induction of pathogenesis. It will then go on to highlight the potential of the schistosome nervous system for drug targeting. Chapter 2 analyzes the identification and classification of putative schistosome GPCRs at the family level. Chapter 3 discusses tissue-specific and pairing dependent GPCR expression in S. mansoni and chapter 4 analyzes schistosome GPCR expression in various life-cycle stages. Chapter 5 investigates the characterization of deorphanized GPCRs in S. mansoni. Lastly, chapter 6 analyzes the pharmacology of schistosome GPCRs.

1. Schistosoma mansoni 1.1 Schistosome eggs play a crucial role in schistosome lifecycle and induction of pathogenesis In contrast to most viruses, protists and bacteria, schistosomes and various other helminths cause chronic infections where parasite and host both live for years. As a result, the parasite is able to maximize its opportunity for transmission and reproduction.20,21 In order to eliminate schistosomiasis, it is fundamental to understand the pathogenesis and life cycle of S. mansoni.

The life cycle of Schistosoma is complex (Figure 1).2 The schistosome eggs are secreted in faeces into the water, and after a motile miracidium has hatched from the egg, it searches for a specific water-snail host, depending on the species. Then, miracidia transform into sporocysts in an intermediate water-snail host.1,22 Subsequently, they reproduces asexually by developing mother and daughter sporocysts. Cercariae are then produced within daughter sporocysts. After approximately four to six weeks, the infectious cercariae leave the snail, move through the water and enter the skin of the human host. Consequently, they lose their tails and become schistosomula. In the human host, the maturing larvae transform into adult male and female schistosomes within the duration of five to seven weeks. The periods in humans and snails in which there is an infection but no release of eggs (humans) or cercariae (snails) can be detected, are called prepatent periods. The schistosomula travel via venous circulation to the lungs and are now called lung schistosomula.23 The schistosomula then travel to the left heart and into circulation. In the liver, the larvae mature and are now called adult schistosomes. Male and female schistosomes form a pair while passing through the liver. As a consequence, the female reproductive organs mature.22 In most species, the paired schistosomes migrate to the mesenteric veins of the gut. However, in S. haematobium the final destination is the urinary venous plexus of the bladder. A matured female can produce 300–3,000 eggs per day, depending on the species. Once the eggs are produced, half of them end up in the gut lumen (most schistosomes) or the bladder (S. haematobium), where they are excreted in faeces or along with the urine. The other half reach the spleen and liver via the blood system. Here they enter the tissues, which causes liver cirrhosis as well as severe inflammation and induces the morbidity. On average, schistosomes live three to ten years in their human hosts, but it is possible for them to live up to forty years.

Figure 1: Lifecycle of Schistosoma. The images represent Schistosoma mansoni.2 Schistosome egg production is not only crucial for life cycle completion but also for inducing the morbidity caused by schistosome infections.18,19 Schistosomiasis is predominantly caused by the immune response of the human host to the eggs of schistosomes.1,18 The eggs of S. mansoni schistosomes that are not excreted end up in the liver and there a granulomatous host immune response is induced. The granulomas contain egg proteolytic enzymes that cause the destruction of the eggs, thereby neutralizing pathogenic antigens. However, they also cause fibrogenesis in the infected tissues.24 The formation of granuloma in the host can be attributed to CD4+ T cell and B cell responses, which are regulated by a variety of cytokines, chemokines and cell populations.21,24– 26 Research has shown that the inherent propensity of antigens in the eggs of schistosomes is the primary reason for the induction of the Th2-type response.25

In summary, schistosome eggs play a fundamental role in schistosome life cycle as well as in the induction of pathogenesis. For this reason, it is crucial to identify and analyze schistosome genes that control reproductive development. Section 3.3 discusses GPCRs that display a gonad preferential expression.

1.2 The nervous system of S. mansoni has great potential for drug targeting As described in the previous section, S. mansoni has a complex life cycle. The nervous system of S. mansoni plays a crucial role in this life cycle and thus in the survival of the parasite. It is involved in attachment, migration, reproduction and feeding.27 During the migration phase, neuronal signaling enables S. mansoni to respond to host cues and is involved in navigating the migration and controlling the somatic muscles.28 The central nervous system enables the suckers of the worm to attach to the host. The nervous system is also used to control the muscle lining of the viscera, including digestive, excretory, and reproductive tracts. Moreover, the central nervous system is involved in the tight coupling of female and male schistosomes. When any of these activities is disrupted through pharmacological intervention, it could lead to interference of the normal life cycle. As a consequence, the worms may be eliminated from the host.

The flatworm nervous system is composed of a simple brain in the head region and multiple pairs of longitudinal nerve cords. The latter are cross-linked by transverse commissures, which results in an orthogonal pattern.29 The central nervous system is connected to a peripheral network of nerve plexuses and nerve cords that innervate the major structures of the worm’s body such as the somatic musculature, the suckers and the tegument. The subtegumental nerve plexuses that innervate the body wall musculature coordinate the activity of various types of fibers, which results in controlled movement. There are diagonal, circular and longitudinal muscle fibers in the body wall.29 These allow the worm to bend, lengthen and shorten.

Since schistosomes do not have a circulatory system, they lack the capability for classical endocrine signaling. Therefore, it is suggested that the nervous system is involved in the signal transduction via paracrine and synaptic mechanisms. Neuroactive compounds bind to their receptors, which results in a direct effect or an effect via second messenger cascades.30 The first class of receptors that are involved are the cysteine-loop ligand-gated ion channels, and the second class consists of GPCRs. These GPCRs have strong potential for drug targeting. In particular, those that are involved in the control of muscle function and movement.31

1.3 Summary In summary, the central nervous system of S. mansoni plays a crucial role in the survival of the parasite. In particular, it is involved in the control of muscle function and movement. Therefore, GPCRs that are expressed in the central nervous system of S. mansoni have strong potential for antischistosomal drug targeting. Furthermore, the pathology that is associated with schistosomiasis is caused almost entirely by schistosome eggs. Therefore, GPCRs that are involved in the reproductions system of S. mansoni are potential drug targets as well. The next chapter analyzes GPCR (sub)families in S. mansoni.

2. GPCR (sub)families in Schistosoma mansoni G protein-coupled receptors are well-established drug targets. All GPCRs have a seven- transmembrane (TM) α-helical structure with the N-terminus being extracellular and the C- terminus intracellular (Figure 2).13,3213 The receptor undergoes conformational changes when an agonist binds, which leads to heterotrimeric G proteins being coupled and activated (Figure 3).32 GPCRs play a role in a wide range of physiological processes. The primary sequence varies significantly among GPCRs. There are two GPCR classification systems that are predominantly used. Firstly, the GRAFS system divides GPCRs into protein families that share a common evolutionary origin.33 These families are Rhodopsin, Frizzled/taste2, Glutamate, Secretin and Adhesion. The second classification system is the A–F system. This system divides GPCRs in invertebrates as well as vertebrates and is primarily based on functional similarities and amino acid sequences.34,35 The GPCRs are divided into classes named class A (rhodopsin-like receptors), class B (secretin/adhesion receptors), class C (glutamate receptors) and class F (frizzled receptors). The two systems mostly differ in the further division of class B into the Adhesion family and the Secretin family in the GRAFS system. This is based on the finding that they do not share a common evolutionary origin. In addition to these families/classes, some organisms have lineage-specific receptors that are part of distinct GPCR families. Since GPCRs modulate a wide variety of critically important physiological and biochemical pathways, they have great potential as therapeutic targets for disrupting crictical proliferation and survival activaties of S. mansoni.15 Therefore, it is important to identify and analyze the GPCRs in S. mansoni. In this section, the GPCR (sub)families that are present in S. mansoni are analyzed.

Figure 2: Structure and topology of GPCRs. A) GPCRs have seven transmembrane helices (gray), three intracellular loops (ICLs), a carboxyl terminus (purple) and three extracellular loops (ECLs) and an amino terminus (orange). The transmembrane domain consists of the transmembrane helices, as well as the extracellular and intracellular loops. B) Cartoon representation of the β2 adrenergic receptor in which the transmembrane helices (TM), loops, and terminal tails are highlighted.32

Figure 3: GPCR activation and signaling. A) An orthosteric ligand (orange) binds an inactive GPCR (example: the β2 adrenergic receptor). B) Subsequently, the GPCR, which is bound to a ligand, undergoes a conformational change to its active state. C) The active GPCR is able to bind a G protein, which then promotes nucleotide release from, and the activation of, the G protein α- subunit.32

2.1 All major GPCR families are present in S. mansoni In order to find anthelmintic drug targets, a deeper understanding of the biology of parasites is crucial. Whole genome sequence data aids in the search for potential receptors. Analysis of the genome of S. mansoni showed that it encodes a minimal of 11,809 genes.36 In 2011, an updated genome of S. mansoni was described.37 More than 45% of the predicted genes were modified and the total number of genes was reduced to 10,852. The first draft of the genome of S. mansoni provided a starting point for the identification and analysis of GPCRs in S. mansoni. Phylogenetic analysis of putative receptors was performed to divide GPCRs in S. mansoni into GRAFS families.15 Various bioinformatic methods were used, including family-specific profile hidden Markov models (HMMs), motif-driven queries and homology-based searching (BLAST).38 A machine-learning approach was used for ligand-based (sub)classification of full-length Rhodopsin GPCRs. The results of the phylogenetic analysis showed that at least 117 GPCR genes and all major GPCR families are present in S. mansoni (Table 1).15 In addition, it revealed a Rhodopsin- like orphan family (PROF1) that is specific to Platyhelminthes.

A more recent study presented an updated phylogenetic analysis of GPCRs in the genome of S. mansoni.17 Every receptor that was used in this study is connected to a gene model that was validated by previously conducted whole transcriptome RNA sequencing (RNA-seq) experiments.37 This updated study found 115 putative GPCRs with at least three predicted transmembrane domains (TMs). 105 of these GPCRs had more than four TMs and were analyzed phylogenetically (Figure 4).17 Subclassifications shown in this figure each correspond to a highly supported node. Each gene ID has a color depending on transcriptomic enrichment. A note of caution is due here since gene IDs can differ per study. For example, they differ between the study conducted by Lu et al. 2016,22 and Hahnel et al. 2018.17 For a comparison between the gene IDs in these two studies, consult supplementary table 1 provided by Hahnel et al. 2018.17 The main difference between the findings in Table 1 and Figure 4 is that the updated study (Figure 4) found less class A GPCRs and a single receptor more in class B as well as in class C. The number of class F receptors is the same in both studies. Figure 4 shows that class A aminergic receptors, including opsins, biogenic amines and orphan amines share a common peptide receptor-like ancestor.

This study phylogenetically analyzed 105 putative GPCR genes that had more than four TMs. In total, 67 seven-transmembrane receptors were identified. All GPCRs that have been deorphanized so far (Chapter 5), contain seven TMs. This raises the question whether putative S. mansoni GPCRs with less than seven TMs are functional. In the following section, the GPCR families that are present in S. mansoni are analyzed. The first group that will be discussed is the Rhodopsin family.

Table 1: Comparison of GPCR repertoires in H. sapiens and S. mansoni based on the GRAFS system.15 H. sapiens S. mansoni R AMIN (α) 44 24 MEC (α) 22 0 MTN (α) 3 0 OPN (α) 11 4 PTGER (α) 13 0 PEP (β) 43 36 CHEM () 43 0 MCHR () 1 0 SOG () 10 0 LGR( ) 7 0 MRG () 7 0 OLF () 494 0 PUR () 35 0 PROF1 0 19 Unclassified 20 22 F FZD/SMO 11 5 TAS2 13 0 G GLR 24 2 A/S ADH 27 3 SEC 20 2 PARF1 0 0

Figure 4: Phylogenetically analyzed S. mansoni GPCR genes. Putative GPCRs were analyzed with MrBayes3.2 to infer a Bayesian tree.39 It is rooted between class A and classes, F, B, and C. Subclassifications are shown and they each correspond to a highly supported node. Each gene ID has a color depending on transcriptomic enrichment. sF (pairing-inexperienced females), bF (pairing-experienced females), bM (pairing-experienced males), sM (pairing-inexperienced males), bT (testes from bM), sT (testes from sM). PROF (Platyhelminth-Specific Rhodopsin-like Orphan-Family), FLPR (FMRFamide-like Peptide GPCRs).17

2.2 Two main groups of the Rhodopsin family GPCRs are present in S. mansoni The four main groups of the Rhodopsin family (α, β, , ) are subdivided into thirteen subclasses in mammalian genomes.40 Group α and β are present in S. mansoni while  and  are absent (Table 1).15 The α group includes opsin-like, amine and melatonin receptors. The amine subclass is largest for both humans and S. mansoni, with a minimum of 44 and 24 putative aminergic receptors, respectively (Table 1).15 Biogenic amines such as histamine, dopamine and serotonin regulate core activities like metabolism, transport and movement in flatworms.41 These activities are crucial for the survival of the organism, and therefore the corresponding receptors are interesting potential drug targets. The other subclass that is present in S. mansoni are the melanopsin-like receptors. Four potential receptors belonging to this subclass were identified. Remarkably, no melatonin-like receptors were identified in S. mansoni, while 9 of such receptors are present in S. mediterranea. 14

Melatonin has been suggested to have regeneration effect in planaria.42 Not all receptors could be classified.15 Many of these 22 unclassified receptors in S. mansoni are likely to be specific to Platyhelminthes.

The β subgroup houses the greater part of peptide and peptide hormone GPCRs. Multiple studies have shown that neuropeptidergic signaling plays an important role in feeding, locomotion, reproduction, regeneration and host-finding.43 36 putative peptide receptors were identified in S. mansoni (Table 1).15 Most of these receptors have no assigned ligand. However, a part of the receptors showed moderate homology to previously characterized FMRFamide-like Peptide (FLP) receptors and neuropeptide F-like (NPF) receptors.44,45 The putative peptidergic receptors are divided into three clades with high posterior probabilities (Figure 4).17 The first clade includes GPCRs that show similarity to Neuropeptide FF (NPFF), Neuropeptide F (NPF) and Neuropeptide Y (NPY). The second clade houses GPCRs that are similar to FMRFamide-like Peptide GPCRs (FLPRs). The third clade is platyhelminth-specific and contains receptors that are members of the Platyhelminth-specific Rhodopsin-like Orphan-Family (PROF). This clade is discussed in the next section.

2.3 Platyhelminth-specific Rhodopsin-like orphan family GPCRs are a major area of interest within the field of helminth neurochemistry and identification of novel targets for antischistosomal drugs.15,28 However, high conservation of GPCRs between the human hosts and the parasites can be a source of selectivity problems. Molecules that target multiple GPCRs may cause toxic side effects.46 A possible solution to this problem is focusing on phylum-specific proteins, in particular orphan proteins. Recently, a highly-diverged Platyhelminth- specific Rhodopsin-like Orphan Family was discovered.15 Platyhelminth Rhodopsin Orphan Family 1 (PROF1) is the largest subfamily because of an expansion in trematodes.47 Other subfamilies are called PROF2 PROF3a PROF3b. No readily identifiably similarity to non- flatworm GPCRs was found.15 It is predicted that PROF1 receptors are solely derived from intronless genes and have a 7 TM domain with an extracellular N-terminus.15 Although the receptors show remnants of ubiquitous Rhodopsin family motifs, no homology was found to previously characterized GPCRs. In S. mansoni, 19 PROF1 receptors were found (Table 1) and in platyhelminth S. mediterranea, 47 receptors were identified. Maximum parsimony analysis of

PROF1 GPCRs made it possible to subdivide the receptors into three subfamilies named PROF1 I, II and III (Figure 5).15 Group I contains the most receptors, including 29 members from S. mediterranea and 13 members from S. mansoni. There are no clear orthologs between species, which suggest that contraction or expansion of the receptors happened after the splitting of trematodes and planaria in the flatworm lineage. Group II houses 12 S. mediterranea and 6 S. mansoni sequences. Group III includes 6 S. mediterranea sequences.

To study transcript expression for putative PROF1 receptors, RT-PCR was utilized.15 Planarian or schistosome tissues were used to extract total RNA from. The putative receptors that were selected are marked with an asterisk in the maximum parsimony tree (Figure 5).15 The two schistosome PROF1 sequences that were used are Smp_084270 (group I) and Smp_041880 (group II). The results confirmed transcript expression of the two schistosome and thirteen S. mediterranea targets in the adult stage. Life stage-specific expression data from S. mansoni was utilized to create expression profiles for PROF genes.37,47 The analysis of these profiles is discussed in section 4.4. Another study discovered that Smp_041880 is predominantly described in the ovary of S. mansoni (section 3.3).17 Since it is grouped into the PROF1 family, it is suggested that natural ligands of this GPCR are flatworm-specific molecules.17 Together, these findings highlight that this receptor is an interesting potential parasite-selective drug target. Drugs that target this receptor could potentially disrupt reproduction processes in S. mansoni.

Research to date has not yet assigned putative ligands or functions to the PROF1 family. It is suggested that members of this family may respond to phylum-specific peptide ligands. Due to the fact that the receptors are highly diverged, determining receptor function will prove challenging. However, the receptors form one of the largest Rhodopsin-like subfamilies that are conserved between the monophyletic species S. mansoni and S. mediterranea. Therefore, the receptors are suggested to play a prominent biological role in both species.

Figure 5: Phylogenetically analyzed PROF1 GPCR genes. A maximum parsimony tree was inferred for all PROF1 receptors that are identified. TM domains I-IV were included in an alignment block, which was bootstrapped 1000 times for parsimony analysis. Three subfamilies were identified for the PROF1 family (I, II, III). Sequences from Schmidtea (blue) and S. mansoni (green) were analyzed. The parsimony tree is rooted with S. mansoni opsin-like GPCR (AAF73286.1). The putative receptors that were selected for RT-PCR analysis are marked (*).15

HMMs that were trained against the originally published S. mediterranea and S. mansoni GPCR complements, were used to identify GPCRs in the genome of F. hepatica.17 In total, six putative PROF1 receptors were identified. This result further supports the notion that the PROF1 receptors are expanded within flatworm lineages. Some studies suggested that the PROF family is similar to the nematode Srw family, which is an ancient chemoreceptor family.48,49 However, while the Caenorhabditis elegans Srw family is primarily (90%) located on one chromosome,50 the S. mansoni PROF orthologs are widely distributed throughout the genome.17

The Platyhelminth-specific Rhodopsin subfamily (PROF) is a lineage specific novel receptor grouping with large numerical representation. Further research into this family may reveal functions or ligands that are specific to Platyhelminthes. Moreover, these receptors are interesting to investigate as selective antischistosomal drug targets. In the next section, the Adhesion and Secretin GPCR Families in S. mansoni are analyzed.

2.4 Six Adhesion/Secretin GPCRs are identified in S. mansoni Although adhesion and secretin GPCRs have similar sequences in their 7 TM domains, they differ in their N-terminal ectodomains.15 Usually, Adhesion receptors contain a long serine and threonine- rich N-terminus that has a variety of functional domains. Deorphanization of a couple of adhesion receptors linked them to a wide variety of ligands such as FMRFamide-like neuropeptides, transmembrane glycoproteins, collagen and complement proteins.51,52 It is hypothesized that vertebrate adhesion GPCRs’ functions vary widely. Mammalian adhesion GPCRs are implicated in controlling cellular migration. In the vertebrate lineage this family is subdivided into eight subfamilies. In total, three adhesion receptors were identified in S. mansoni.15 One of these receptors (Smp_099670) contains a Somatomedin B domain and another adhesion-like receptor was found (Smp_058380) that contains an N-terminal GPS domain.15

An archetypical secretin GPCR contains a hormone-binding domain (HBD) in the N-terminus that interacts with peptide hormones.53 Two GPCRs of this family were found in S. mansoni. One of these receptors showed homology to diuretic hormone receptors and is likely to be involved in homeostatic regulation (SMP125420). The other secretin receptor was found to have a hormone receptor domain (HRM) domain and showed sequence similarity to parathyroid hormone receptors (SMP170560). The aforementioned updated study,17 identified an additional adhesion/secretin (class B) receptor (Smp_125420).

2.5 S. mansoni houses three putative glutamate-like GPCRs Glutamate GPCRs respond to a wide variety of endogenous agonists such as γ-aminobutyric acid (GABA), glutamate, odorants and calcium ions.15 There are eight mammalian metabotropic glutamate receptors (mGluRs) types that are split into three subgroups based on G-protein coupling properties, agonist pharmacology and amino acid sequence similarity.54 They all contain a large extracellular domain that has a ligand binding domain (LBD).

Two glutamate-like receptors were found in S. mansoni. The previously mentioned updated study,17 identified an additional glutamate (class C) receptor. One glutamate-like receptor (GSmp_052660) showed homology with a Drosophila mGluRlike receptor (DmGluRA).15 However, the second receptor (GSmp_128940) seemed to be significantly diverged from mGluR as well as from GABAB receptors. The characterization of this receptor is discussed in section 5.1. Figure 6 shows a comparison between conserved mouse mGluR3 LBD residues that take part in glutamate binding with Glutamate-like receptors GSmp_128940 (S. mansoni) and GSMD004608 (Schmidtea mediterranea).15 This analysis showed that GSMD004608 has retained key residues that interact with glutamate α-amino and α-carboxylic groups. Furthermore, it shows that GSmp_128940 has an unusual LBD with only one putative glutamate-interacting residue. This receptor displays overall divergence with the canonical glutamate binding pocket and most likely have atypical pharmacology or responds to different amino acid-derived ligands. The pharmacological profile of Smp_128940 is analyzed in section 6.1.

Figure 6: Comparison between conserved mouse mGluR3 LBD residues that take part in glutamate binding (underlined) with Glutamate-like receptor GSmp128940/SmGluR (S. mansoni) and GSmd004608 (Schmidtea mediterranea). The residue locations are shown in numbers (mouse mGluR3 sequence) and disagreements in positions are shown in red.15

2.6 Five S. mansoni GPCRs are identified that are part of the Frizzled Family GPCRs in the Frizzled family usually contain a cysteine-rich ligand-binding domain and seven hydrophobic domains.55 The endogenous ligands are, among others, secreted Wnt proteins. The receptors play a critical role in developmental processes such as cell motility, synaptic organization, cell polarity and cell fate determination. Moreover, in planarian flatworms the canonical Wnt/b-catenin pathway is involved in regeneration polarity.56,57 Four Frizzled sequences and one Smoothend-like sequence were identified in S. mansoni.15 In humans, ten Frizzled GPCRs are phylogenetically grouped into four clusters. A part of the S. mansoni GPCRs could also be grouped into these clusters. A single Frizzled GPCR in S. mansoni grouped in cluster IV (FSmp_118970), which shares ~38% amino acid identity with human Fzd6 and ~45% with Drosophila Fzd1. Two receptors grouped in cluster II (FSmp_155340 and FSmp_139180). The remaining receptors could not be grouped.

2.7 Summary This chapter analyzed the identification and classification of putative schistosome GPCRs at the family level. The most recent study based on whole genome sequencing data has described the S. mansoni genome with 10,852 genes.37 Based on these data, 115 putative GPCRs were found with a minimum of three predicted TMs. 105 of these GPCRs had more than four TMs and were analyzed phylogenetically. A previously conducted phylogenetic analysis described 117 GPCR genes that belonged to five major GPCR families.15 These include the Glutamate family (2 GPCRs), Rhodopsin family (105 GPCRs), Adhesion family (3 GPCRs), Frizzled family (5 GPCRs) and the Secretin family (2 GPCRs). The updated study reduced the number of GPCRs in the Rhodopsin family (class A), added one receptor to the Secretin/Adhesion family (class B) and one to the Glutamate family (class C).37 The majority of the GPCRs that were identified belonged to the α or β Rhodopsin subgroup. The identification of these receptors may help identify biogenic amine and neuropeptide-like ligands in S. mansoni. The most striking result is the identification of a new and highly-diverged receptor family named Platyhelminth Rhodopsin Orphan Family 1. The members of this family (19 GPCRs) share little sequence similarity with any previously discovered GPCR. For this reason, they have enormous appeal as potential drug targets for antischistosomal drugs with high selectivity for S. mansoni GPCRs over those of the mammalian host.

The identification of S. mansoni GPCRs provides opportunities to identify receptors that are fundamental for parasitic platyhelminth life and survival. These are potential drug targets in schistosomes. The identified GPCRs are potential candidates for future functional and biological studies. The next chapter analyzes the tissue-specific and pairing dependent GPCR expression in S. mansoni.

3. Tissue-specific and pairing dependent GPCR expression in adult S. mansoni

3.1 Analysis of tissue-specific GPCR expression in paired and unpaired schistosomes The reproductive biology of schistosomes is unique, as they are the only one to have evolved separate sexes among trematodes.58 The reproduction of schistosomes involves a high degree of interdependence between females and males. Adult females and males live paired, which is necessary female gonad development.59 Pairing-inexperienced females (sF) exhibit an immature reproductive system. The vitellaria is underdeveloped and the ovary is small and contains stem cell-like precursor oocytes. The maturation of the vitellarium and ovary is induced when the female pairs with the male schistosome, leading to a sexually mature female (bF). Contrary to females, pairing-inexperienced males (sM) have testes with differentiated spermatocytes and are not morphologically different from pairing-experienced males (bM).60,61 Nonetheless, sM and bM gene expression differ as a result of pairing.22,62 In this chapter, the tissue-specific and pairing dependent GPCR expression in S. mansoni is analyzed. This can provide new insights into GPCR functions. Moreover, the data can provide a selection of GPCR candidates for functional studies.

Figure 7: Female and male worm paired.63

Whole-organ isolation can be used to analyze specific tissues in S. mansoni.64,65 Based on this method, a tissue-specific comparative RNA-seq analysis was conducted on complete testes and ovaries of males and females from paired and unpaired schistosomes.22 In adult schistosomes, most GPCRs detected in the RNA-seq analysis exhibited tissue-specific and/or sex-/pairing-dependent transcription. For this reason, GPCRs can be categorized into functional groups (Figure 8).17

Figure 8: Hierarchical clustered GPCR genes that are expressed in adult S. mansoni. A light color indicates a relative low expression level, whereas a darker color indicates a relative high expression (Color Key). bF (pairing-experienced females), sF (pairing-inexperienced females), sM (pairing-inexperienced males), bM (pairing-experienced males), bT (testes from bM), sT (testes from sM), bO (ovaries from bF), sO (ovaries from sF).17

3.2 S. mansoni GPCRs contribute to non-gonad, pairing-dependent processes The majority of GPCRs that were found during the RNA-seq analysis are predominantly expressed in non-gonad tissues of both male and female schistosomes.17 For example, the dopamine- responsive GPCR SmD2 (Smp_127310) is located in the acetabulum and the subtegumental somatic musculature of all larval stages.66 In adult schistosomes, the receptor was mainly present in the somatic muscles and in a lower amount in the muscular lining of the caecum. The results of functional assays showed that that SmD2 responded dose-dependently to dopamine (section 5.3).

It is suggested that this receptor plays an important role in the neuromuscular system of S. mansoni. A second receptor that is mostly expressed in non-gonad tissues is the serotonin receptor Sm5HTR (Smp_126730). Immunolocalization of the receptor showed that it is widely expressed in the nervous system of adult worms and schistosomula.31 Specifically, it is present in the main nerve cords and the cerebral ganglia. Furthermore, the receptor is expressed in the tegument and the peripheral innervation of the body wall muscles. In section 5.5, a functional analysis of Sm5HTR is described.

Confocal immunolocalization studies showed that glutamate receptor SmGluR (Smp_128940, section 5.1) is widely expressed in the central nervous system of both adult schistosomes and larvae.67 No expression was measured in the vitellarium and ovary. In adult schistosomes, the receptor is located in the cerebral commissures and the main longitudinal nerve cords. Moreover, it is expressed in the peripheral nerve plexuses and fibers that innervate the body wall musculature and acetabulum. In female schistosomes, SmGluR was located at the uterus, ootype and oviduct. Since SmGluR is expressed in the main nerve cords, it may be involved in interneuronal signaling. In other organisms, mGluRs are also often involved in signaling within the central nervous system. In vertebrates, they are located presynaptically where they modulate transmitter release, or post- synaptically to mediate excitatory responses. The authors note that distribution of SmGluR resembles that of acetylcholine and the peptidergic system in central nervous system of schistosomes. This suggests that the receptor could play a role in the control of one or more of these circuits.29

Interestingly, most of the GPCRs that are predominantly expressed in non-gonad tissue exhibited a pairing-dependent transcription. This is in agreement with recent studies that revealed that neuronal processes are important in S. mansoni male-female interaction.22,68 Furthermore, most of the non-gonad tissue GPCRs belong to the sM-bM-sF group (Figure 8).17 This is consistent with the finding that the sF transcriptome is more similar to the male than to the bF.22 This observation suggest that biological processes such as partner attraction and locomotion are equally important in sF, sM and bM. About 30% of the receptors within this group showed a bias towards one of the three subgroups. Receptors that showed a bias towards sM may play a role in female attraction or locomotion. Similarly, GPCRs that showed a bias towards sF may be involved in male attraction

24 or repression of sexual maturation. Previous microarray studies showed that transcript levels of genes that are involved in egg production increase in bF compared to sF.22,62 In addition, transcription levels of genes that are associated with motility are higher in sF than in bF. In contrast, these levels are increased in bM compared to sM. This also supports the suggestion that the transcription levels of GPCRs are increased in bM to increase muscle activity, which is necessary to successfully transport the female.17 In addition, GPCRs that are preferentially transcribed in bM might be associated with clasping and pairing processes.

This section analyzed the expression of GPCRs in non-gonad tissue of female and male schistosomes. The results revealed that GPCRs are involved in non-gonad, pairing-dependent processes in S. mansoni. These potentially mediate functions that are not related to the gonads during the schistosome male-female interaction. The next section analyzes GPCR expression in the gonads of schistosomes.

3.3 S. mansoni GPCRs play a role in gonad-specific functions As discussed in section 1.1, schistosome eggs play a fundamental role in schistosome life cycle and in the induction of pathogenesis.22 Therefore, understanding the processes that control egg production and gonad development is essential for discovering strategies to eliminate schistosomiasis.69 In this section, GPCRs that are preferentially transcribed in the gonads of S. mansoni are discussed.

The schistosome female undergoes sexual maturation upon pairing, which is marked by a change in gene expression. Remarkably, only a few GPCRs exhibited gonad-biased transcription (Figure 8).17 A previous study has demonstrated that transcription levels of genes that are associated with egg production are increased in bF compared to sF.22,62 Four S. mansoni GPCRs are preferentially transcribed in bF (Smp_041880, Smp_049330, Smp_170560 and Smp_145240). All of these are functionally uncharacterized. These receptors are abundantly expressed in the vitellarium, since this is the most prevalent tissue in this group.17,59,70 The vitellarium plays an important part in the synthesis of composite eggs. Drugs that target these receptors may disturb the reproduction process of S. mansoni. Smp_041880 is the only one that is preferentially transcribed in the ovary.17 This putative receptor is an ortholog of a potential allatostatin receptor, which is involved in the

25 reproductive development in female S. japonicum.71 Allatostatin family neuropeptides are crustacean and insect hormones that play a role in feeding, reproduction and the generation of juvenile hormone.72 However, Smp_041880 is grouped into the PROF1 family (section 2.3), which suggests that natural ligands of this GPCR are flatworm-specific molecules.17 Smp_145240 was phylogenetically grouped in the FLPR clade, while Smp_170560 belonged to the Adhesion/Secretin family (class B) (Figure 4).17 The last receptor in the bF group could not be classified (Smp_049330). Smp_174350 is also preferentially expressed in the female gonads. It is a frizzled ortholog and its expression levels are increased in the ovary of sF (Figure 8).17 It is hypothesized that it is involved in a Wnt signaling pathway and preserving the immature state of the oocytes.59,61

A few GPCRs are preferentially transcribed in the male reproductive organs. Seven GPCRs are preferentially transcribed in the testis (Figure 8; sT-bT group).17 These include Smp_178420 and Smp_127720, which are phylogenetically classified as class A orphan amine and biogenic amine receptors, respectively (Figure 4).17 Smp_244240 and Smp_242910 are also part of the sT-bT group and are connected to neuropeptide signaling. Interestingly, a recent study showed that neuropeptides play an important role in planarian germline development.44 The remaining GPCRs in the sT-bT group are classified as class B (secretin/adhesion) GPCRs (Smp_099670, Smp_176820) and as an opsin receptor (Smp_104210). Notably, the transcription levels of four GPCRs in the sT-bT group increased in the testis upon pairing. In accordance with this result, previous studies have demonstrated that changes occur pairing-dependently in the schistosome testis.22,65,68 Possibly, one of these changes is the increase of sperm production upon pairing.

3.4 Summary In summary, the results in this chapter showed that there are GPCRs that are preferentially transcribed in the gonads of S. mansoni. These GPCRs may contribute to embryogenesis, vitellogenesis and/or gametogenesis. Furthermore, most GPCRs have a non-gonad-preferential, pairing-dependent transcript profile. These GPCRs may play a role in the interaction between male and female schistosomes. Since schistosome eggs play a critical role in schistosome life-cycle and in the induction of pathogenesis, identifying receptors that mediate egg production and gonad development will help in the identification of new drug targets in S. mansoni. Similarly, the male- female interaction is crucial for the induction of germ-cell differentiation. GPCRs that play a role in this interaction are interesting potential candidates for antiparasitic drug targeting. The following chapter analyzes GPCR expression in various S. mansoni life-cycle stages.

4. GPCR expression in various S. mansoni life-cycle stages As described in section 1.1, S. mansoni goes through many different and complex life stages. Therefore, it is expected that the expression of the GPCRome differs per life stage. Since juvenile schistosomes are refractory to the currently available drug (PZQ) to treat Schistosomiasis,8 it is important to develop drugs that are effective against this life stage. The molecular target of PZQ remains unknown. However, it is known that downstream factors play a role in PZQ action. Similar to adult S. mansoni, schistosomula that are refractory to PZQ experience Ca2+- dependent paralysis and contraction.73 However, whereas adults worms do not survive, juvenile worms recover and live. This indicates that the initial target is probably similar, but adaptive responses that result in the survival of the animals are activated in juvenile, and not adult, worms.9 This example illustrates that gene expression patterns differ per S. mansoni life stage. Understanding the link between the S. mansoni GPCRome and the various life stages will help to find new drug targets. This chapter examines the differences in GPCR expression in various S. mansoni developmental stages. Firstly, GPCR expression in adult schistosomes compared to that of other life-stages is described. Secondly, the developmental expression pattern of biogenic amine receptors (SmGPR-1, SmGPR- 2) is analyzed. Then, this developmental expression pattern is compared to that of glutamate receptor SmGluR in order to gain insight into the changes in the larval nervous system during its development. Lastly, the expression of PROF complements during various life stages is analyzed.

4.1 GPCRs are low-abundantly expressed in adult schistosomes A tissue-specific comparative RNA-seq analysis was conducted by Lu et al. on complete testes and ovaries of males and females from paired and unpaired schistosomes.22 Data obtained from this study showed that, compared to the GPCRome described by Hahnel et al.,17 60% of the GPCR genes were expressed in adult schistosomes. Moreover, it revealed that GPCRs of all classes were present. A possible explanation for the fact that not all GPCR genes are expressed, or are weakly expressed, in adult schistosomes is that the missing GPCRs could be predominantly important for earlier life stages. For example, the need for miracidia to quickly find a host without losing their infectivity illustrates that miracidia need highly adapted sensory mechanisms.74 A previous RNA- seq study that investigated GPCR genes in miracidia showed that Smp_180030 was present in light-responsive miracidia.74 This study suggested that this gene together with Smp_104210 is involved in photokinesis and plays a fundamental role in host-finding. Both were predicted as

28 opsin-like GPCRs. Interestingly, only Smp_104210 was detected in adult schistosomes and exhibited a testis-biased transcription.17 Seven other genes were found to be expressed in free- living miracidia, each sequence containing 7 TM domains. These include Smp_173010, which has been described as a PROF receptor.75

Analysis of the data from a previously conducted RNA-seq study,37 showed that the majority of the 47 putative S. mansoni GPCRs that were missing in the Lu et al. dataset are less abundantly transcribed in adult schistosomes in comparison to other life stages (Figure 9).17 This explains their absence in the Lu et al. dataset. Furthermore, this dataset did not include other GPCRs that were previously characterized, but were transcribed below threshold.22 These included the amine receptors SmGPR-1 (Smp_043260), SmGPR-2 (Smp_043340) and SmGPR-3 (Smp_043290). The next section discusses the gene expression patterns of two of these biogenic amine GPCRs.

Figure 9: Analysis of the expression of the 47 putative GPCRs that were missing in the Lu et al. dataset per S. mansoni life- cycle stage (highlighted in blue).17,22

4.2 Biogenic amine receptors are upregulated in parasitic stages Biogenic amine receptors SmGPR-1 (Smp_043260), SmGPR-2 (Smp_043340) and SmGPR-3 (Smp_043290) are expressed in the nervous system of adult schistosomes (section 5.4).76–78 SmGPR-1 and SmGPR-2 were both shown to be activated by histamine (section 5.4.3 & 5.4.6),76,79 whereas SmGPR-3 responds to dopamine (section 5.4.8).80 A study focused on the relative transcription levels of SmGPR-1 in various life stages of S. mansoni.77 Quantitative RT-PCR was used to measure expression of SmGPR-1 at the mRNA level. First, the expression levels of adult schistosomes, cercariae and schistosomula were compared (Figure 10A).77 The results suggested that the receptor is upregulated after cercarial transformation. Compared to cercariae, a 10-fold increase (P < 0.01) in expression was measured in transformed schistosomula (S0). These levels were measured again seven days later (S7), which showed that they increased even further (P < 0.0001). Remarkably, the expression levels went down again after 14-days and in adults down to S0 level. Since this upregulation in schistosomula peaks at day 7, it could be hypothesized that histamine signaling plays a role in the initial development of larvae and during the lung-stage, which occurs approximately on 7-8 days post-infection.23,77

A second experiment was performed in which mRNA levels of SmGPR-1 were measured in S. mansoni miracidia and sporocysts (Figure 10B).77 Compared to miracidia, a 20-fold (P < 0.01) increase in mRNA levels was observed in 4-day old sporocysts. Remarkably, a more than 200-fold increase in 20-day old sporocysts (P < 0.001) was observed compared to miracidia. Possibly, the receptor plays a role in the regulation of the growth and development of sporocysts. This significant SmGPR-1 gene upregulation in parasitic stages (sporocysts, schistosomula and adults) in comparison to free swimming stages (miracidia and cercariae) suggests that SmGPR-1 has a fundamental role in the establishment and/or maintenance of parasitism within the hosts.

Figure 10: Developmental expression of SmGPR-1. A) Expression of SmGPR-1 relative to cercariae. Quantitative RT-PCR was performed on reverse-transcribed RNA obtained from S. mansoni cercariae, schistosomula up to 14 days after transformation (S14) and adult schistosomes. B) Expression of SmGPR-1 relative to miracidia. Data obtained from qPCR on reverse-transcribed RNA obtained from miracidia and sporocysts (4-day and 20-day).77

Structurally related to SmGPR-1 is SmGPR-2 (Smp_043340).76 To establish whether the expression of SmGPR-2 is also developmentally regulated, real-time qPCR was used to compare mRNA levels in various S. mansoni developmental stages. Similar to SmGPR-1, an increase in expression level was observed immediately after cercariae transformed to stage 0 schistosomula (S0) (Figure 11). This level increased up to 60-fold at day 7 (P < 0.001), after which the expression levels decreased. In adult schistosomes, the expression level is similar to S0 level. The SmGPR-1 and SmGPR-2 developmental patterns are highly comparable,77 which suggest that these kind of receptors are especially important during the development of early schistosomula.

Figure 11: Developmental expression of SmGPR-2 in S. mansoni. Expression of SmGPR-2 relative to cercariae. qPCR was performed on reverse-transcribed RNA from cercariae (C), schistosomula up to 14 days after transformation (S14), and adult schistosomes (A).76

The mRNA levels of SmGPR-1 and SmGPR-2 are lower in adults compared to other life stages. This might be an explanation for that these receptors were missing, due to transcription levels below threshold, in the transcriptome data obtained by the study conducted by Lu et al.22 The described expression pattern could be a result of general upregulation of neuronal genes in young schistosomula compared to other life stages. Another explanation could be that the upregulation is linked to some part of histamine signaling that happens early in development. Previous research has suggested that young schistosomula increase vascular permeability by using the host’s histamine system.81–83 This facilitates passage through blood vessels during larval migration. It might be the case that the upregulation of S. mansoni’s own histamine system is involved in this.

4.3 The larval nervous system undergoes broad changes during its development Similar to SmGPR-1 and SmGPR-2, SmGluR (Smp_128940) is widely expressed in the nervous system of S. mansoni (section 3.2).67 SmGluR was shown to be activated by glutamate (section 5.1). Remarkably, the expression pattern of up- and down-regulation that was observed for SmGPR-1 and SmGPR2,76,77 was also described for glutamate receptor SmGluR.67 Real-time

32 quantitative RT-PCR was used to determine the expression of SmGluR at mRNA level in various life stages of S. mansoni. The results indicated that expression levels of the receptor are similar for adult schistosomes and cercariae. Remarkably, a significant 8-fold upregulation of SmGluR was found in 8-day old schistosomula compared to cercariae. These findings suggest that the receptor is upregulated during the development of early schistosomula, followed by down-regulation during the transformation from larvae into adult worms. The expression pattern of up- and down- regulation for these three S. mansoni neurotransmitter receptors, SmGPR-1, SmGPR-2 and SmGluR, suggests that the larval nervous system undergoes broad changes during its development. The next section discusses the developmental expression profiles for PROF genes.

4.4 Increased expression of PROF complements during juvenile S. mansoni life stages As explained in in section 2.3, PROF1 is a new highly diverged platyhelminth specific GPCR subfamily.15 The receptors that within this family are interesting because they are exclusively expressed in flatworms, and could possibly function as specific targets for anthelmintics. Since PZQ lacks efficacy against juvenile S. mansoni, it is desirable that new antischistosomal drugs are effective against various life stages.8 Therefore, this section analyzes the expression of PROF complements during various life stages. Life stage-specific expression data from S. mansoni was utilized to create expression profiles for PROF genes (Figure 12).37,47 The results were normalized, allowing stage-to-stage comparison. The results showed that there is a general pattern of increased expression in the juvenile schistosomula life stage. Remarkably, this is upregulation in schistosomula was also observed for SmGPR-1, SmGPR2 and SmGluR (section 4.2 & 4.3).

The expression of PROF receptors in parasitic stages and the restriction of these receptors to flatworms make them interesting potential antischistosomal drug targets. To develop a full picture of PROF receptors, additional studies need to investigate their biological function and endogenous ligands.

Figure 12: Normalized expression of PROF complements in S. mansoni during various life-stages.47

4.5 Summary Taken together, the results in this chapter showed that GPCRs in adult schistosomes are generally expressed at low levels.17 In accordance with earlier observations in other organisms that suggested that GPCR transcription levels are lower compared to other genes.84 Biogenic amine receptors SmGPR-1 and SmGPR-2 are upregulated in parasitic stages, which makes them interesting drug targets. These receptors and SmGluR, which are all neurotransmitter receptors, seem to be upregulated during the development of early schistosomula. This is followed by down-regulation during the transformation into adult schistosomes. These results shows that the larval nervous system undergoes broad changes during its development. Furthermore, analysis of expression data from S. mansoni showed that PROF genes are upregulated in schistosomula as well. This is notable, since it is desired that the next antischistosomal drug is effective against schistosomula. Therefore, SmGPR-1, SmGPR-2 and SmGluR are interesting potential drug targets. In the next chapter, deorphanized S. mansoni GPCRs will be discussed.

5. Deorphanized S. mansoni GPCRs Most GPCRs in S. mansoni remain to be deorphanized. The identification of endogenous ligands for schistosome GPCRs is a necessary step towards the functional characterization of GPCRs and helps unravelling the biological meaning of neuronal signaling via GPCRs.85 This, in turn, advances the understanding of the biology of schistosomes and their complex interplay between the sexes. Furthermore, deorphanization of schistosome GPCRs reveals potential candidates for antischistosomal drug targeting. Cloning and functional expression studies are necessary to deorphanize receptors.28 To deorphanize a receptor, the recombinant protein is expressed in an appropriate cell environment, after which various ligands are tested in order to find a selective agonist (Figure 13).28 This can be a challenge for S. mansoni GPCRs, since there are no flatworm cell lines that can be used for transfection. Moreover, it can be problematic to express schistosome cDNAs in heterologous environments. More specifically, it can be challenging to target the GPCR to the cell membrane at sufficient levels so that the activity can be measured. Furthermore, it is often difficult or not possible to predict which ligands are effective based on sequence homology. As a result, a wide variety of ligands and metabolites at various concentrations needs to be tested. Therefore, a highly adaptable functional expression assay is necessary. A recent pilot study described a novel approach to deorphanize S. mansoni receptors.85 This study used the Membrane- Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to detect potential neuropeptide ligands for schistosome GPCRs. The results indicated that this system is suitable to detect S. mansoni GPCR–ligand interactions. An advantage of this method is that the ligands and the receptor are co-expressed at the membrane. This approach may help in the deorphanization of schistosome GPCRs. So far, seven S. mansoni GPCRs have been deorphanized (Figure 14).86 These include SmGluR (section 5.1), SmGAR (section 5.2), SmD2 (section 5.3), SmGPR-1, SmGPR-2 and SmGPR-3 (section 5.4) as well as Sm5HTR (section 5.5). This chapter investigates the characterization of these deorphanized GPCRs in S. mansoni.

Figure 13: Characterization and deorphanization strategies of GPCRs in S. mansoni.28

Figure 14: Deorphanized S. mansoni GPCRs. All receptors have been deorphanized by RNA interference or in heterologous expression systems. The color of the receptor indicates the activity: green (excitatory function) and gray (unknown function).86

5.1 Glutamate receptor SmGluR Glutamate is a well-established neurotransmitter in helminths, both flatworms and nematodes.67 Research has shown that adult S. mansoni muscle fibers contract in response to glutamate in a dose- dependent manner.87 Furthermore, a study provided evidence for the presence of glutamatergic receptors by showing that S. mansoni worms underwent hyperkinesias and body wall contractions after treatment with a glutamate agonist.88 These results suggest that glutamate has neuromuscular activity in S. mansoni and show that glutamate receptors are potential drug targets. The first metabotropic glutamate receptor that was identified in S. mansoni is SmGluR ((G)Smp_128940).67 As discussed in section 4.3, the expression levels of SmGluR in S. mansoni are similar for adults and cercariae. Furthermore, this receptor is upregulated during the development of early schistosomula. This section discusses the functional analysis of SmGluR.

5.1.1 SmGluR is selectively activated by glutamate Analysis of the protein sequence of SmGluR revealed that the receptor has 1026 amino acid residues.67 SmGluR has conserved features of glutamate receptors, but is only distantly related to mGluRs from other phyla. SmGluR cannot be grouped in any subtypes of mGluRs (Groups I, II and III) and therefore seems to be a novel type of glutamate receptor. To functionally analyze the receptor, SmGluR was heterologously expressed in mammalian HEK-293 cells.67 The full-length cDNA was modified using PCR in order to introduce a Kozak sequence to increase translational efficiency in mammalian cells followed by a FLAG fusion tag at N-terminal end of the receptor. Since most mGluRs inhibit adenylyl cyclase and decrease cAMP levels,89 receptor activity was tested by measuring changes in the level of intracellular cAMP. Important to mention is that the cloned SmGluR cDNA that was used in this study differs slightly from the new annotation (Smp_128940) at the 5’ end. The researchers assumed the sequence to be correct because the length of the SmGluR cDNA is in accordance with the size of the SmGluR protein in the Western blot analysis and it was verified in multiple clones. The authors also suggest that there could be multiple forms of SmGluR in S. mansoni.

Protein expression was monitored by probing the clones with an anti-FLAG m2 antibody, after which the cells were treated with a secondary FITC-conjugated anti-mouse antibody. Green fluorescence was observed in the positive clones (Figure 15A), which were picked for functional

39 analysis.67 The results revealed that glutamate activated the receptor and stimulated the cAMP 67 production dose-dependently with an EC50 of ±100 µM (Figure 15C). This suggests that the receptor is coupled to a Gs type protein, which stimulates adenylate cyclase. In contrast, mammalian mGluRs usually couple to Gi/o and decrease the cAMP level or bind to Gq/11 and increase intracellular Ca2+. The unusual signaling of SmGluR could be a result of the heterologous system. Since SmGluR did not respond to GABA or aspartate, it is selectively activated by glutamate (Figure 15B).67

Although SmGluR has conserved features of family C GPCRs, it is distantly related to glutamate receptors from other phyla and therefore it cannot be classified according to the mammalian system. The receptor shares low level of sequence homology (25–30%) with all types of mGluRs (mGluRs I, II, III).67 The receptor has residues in its binding domain that are associated with glutamate binding. To illustrate, residues Arg-323, Tyr-236, Asp-208, and Ser-165 are thought to undergo an interaction with glutamate in the binding pocket of mGluRs.90 These residues are all conserved in the sequence of SmGluR. However, the schistosome GPCR has a lysine in the position of Arg-78. A previous study showed that this arginine interacts with the carboxylate group in the side chain of glutamate, and is crucial for binding of glutamate to mGluR1 with high affinity.91 It may be the case that lysine has a similar function as arginine, or a different site is involved in the interaction between glutamate and SmGluR.

Figure 15: Functional analysis of SmGluR. SmGluR was expressed heterologously in HEK-293 cells. A) Indirect immunofluorescence assays (IFA) were performed by probing the clones with an anti-FLAG m2 antibody, after which the cells were treated with a secondary FITC-conjugated anti mouse antibody. Green fluorescence was observed in the positive clones. B) Positive clones, expressing SmGluR, were treated with either GABA, l-aspartate or l-glutamate (10-4 M or vehicle -). A competitive immunoassay was performed to test for cAMP. Mock transfected cells were used as controls and to normalize the data. C) Experiments were repeated with various concentrations of glutamate. SmGluR is dose-dependently activated by glutamate.67

5.1.2 SmGluR may influence muscle function indirectly As described in section 3.2, SmGluR is widely expressed in the nervous system of S. mansoni as well as in the female reproductive tract. A previous study suggested that glutamate has neuromuscular activity in S. mansoni.88 It is possible that SmGluR is involved in this effect. However, another study suggested that glutamate receptors are not present in isolated muscle fibers of S. mansoni.87 Hence, SmGluR could modulate neuronal circuits and by doing so influence muscle function indirectly. It is also possible that other glutamate receptors are responsible for the observed effect of glutamate on the musculature. In situ immunolocalization analysis also revealed that SmGluR is associated with the epithelium of the reproductive tract or the oocytes, similar to what has been observed for certain mammalian mGluRs.92,93 Glutamate has been suggested to be important for the regulation of egg production and release in mammals. If this is also the case in S. mansoni, glutamate GPCRs could be potential targets for drugs aimed at inhibition of oogenesis. This would be beneficial for the control of schistosomiasis since schistosome eggs play a

41 fundamental role in schistosome life cycle and in the induction of pathogenesis.22

In summary, glutamate receptor SmGluR is selectively activated by glutamate and may be coupled to a Gs type protein, which means that it has a different signaling mechanism than mammalian mGluRs. The receptor potentially influences muscle function indirectly and may be involved in the regulation of egg production and control. However, further work is required to establish the exact role of SmGluR and L-glutamate in S. mansoni. Section 6.1 analyzes the pharmacological profile of SmGluR. The next section investigates the characterization of acetylcholine receptor SmGAR.

5.2 Acetylcholine receptor SmGAR As discussed in section 1.2, the central nervous system of S. mansoni is crucial for the survival of the parasite. In particular, it is involved in the control of muscle function and movement. Schistosomes lack the capability for classical endocrine signaling and therefore it is suggested that the nervous system is involved in the signal transduction via paracrine and synaptic mechanisms.27,94 Neuroactive compounds bind to their receptors, which results in a direct effect or via second messenger cascades.30 One of these neuroactive compounds is acetylcholine (ACh), which is a quaternary amine neurotransmitter. Usually, it plays an excitatory role in vertebrates and invertebrates. However, some studies suggest that it acts as an inhibitory neurotransmitter or modulator in schistosomes. When the S. mansoni acetylcholine receptors are activated, muscular relaxation and flaccid paralysis occurs.95,96 There are two putative muscarinic cholinergic receptors (mAChRs) that might be responsible for this effect. The first one (Smp_152540) is possibly truncated, but the second one does have all structure features of a complete GPCR.37 mAchRs are part of the GPCR superfamily and are structurally related to class A GPCRs. Invertebrate G protein- coupled acetylcholine receptors (GARs) and vertebrate GARs are homologs due to their structural similarity and broad expression patterns. However, their pharmacological profiles differ significantly.97,98 For this reason, and because of their important functional roles, GARs in S. mansoni are promising targets for antischistosomal drugs.94 This section analyzes the first schistosome GAR named SmGAR (Smp_145540). Functional and structural analysis of SmGAR as well as RNAi phenotypic assays are described.

5.2.1 SmGAR is a constitutively active acetylcholine GPCR in S. mansoni The first schistosome GAR that was functionally analyzed is SmGAR.94 SmGAR shares the highest homology with Caenorhabditis elegans GAR-2 (44%), Ascaris suum GAR-like receptor (35%) and a putative GAR-2 receptor from Clonorchis sinensis (42%). The expression of this receptor is predicted to be highly upregulated during the early larval stages of S. mansoni.37 The activity of SmGAR was measured using a yeast functional assay.99 In short, a plasmid that contained the coding sequence for SmGAR was transformed into yeast (S. cerevesiae) that are autotrophic for histidine and a HIS-deficient medium was utilized. Various yeast strains were tested, and a strain that expressed alpha subunit Gαi2 (CY13393) yielded receptor activity. The last five and first 31 codons of the chimeric Gα gene were of human Gαi2 instead of those of native yeast (GPA1). Strains containing Gαq and Gαs yielded no receptor activity. Activation of the heterologously expressed receptor allows for the expression of the HIS3 reporter gene, which is under the control of the FUS1 promoter, via the yeast’s endogenous pheromone response. This in turn, results in yeast growth. Fluorometric redox indicator Alamar Blue is then used to measure the receptor activity by yeast growth.

The results of the functional assay showed that when SmGAR is expressed in yeast, it has high basal activity (Figure 16A).94 This suggests that the receptor exhibits a propensity towards spontaneous activation. Cells expressing SmGAR that were treated with water (vehicle) showed high levels of receptor activity, while cells that were transformed with empty plasmid (mock control) showed no activity. Spontaneous GPCR activation could be a result of overexpression of SmGAR in recombinant systems. However, studies have shown that constitutively active GPCRs are of biological relevance.100 They result in continuous signaling, which potentially has important (patho)physiological consequences. Furthermore, GPCRs that show constitutive activity can often be activated above their elevated baseline when they are treated with an agonist.101

Figure 16: SmGAR is activated by acetylcholine and exhibits constitutive activity. A) SmGAR (black bars) was expressed in yeast cells (S. cerevesiae). Empty plasmids were used as mock controls (red bars). The cells were tested for receptor activity by treating them with test compounds (10-4 M) or water (vehicle). *** P< 0.0001 B) SmGAR can be activated dose-dependently by acetylcholine and C) carbachol. No activity was observed in the mock controls (red squares).94

To test whether SmGAR could be activated above its baseline, yeast cells expressing the receptor were treated with various neuroactive compounds (100 µM). The results indicate that the receptor is significantly activated above the baseline by acetylcholine (P < 0.0001). In contrast, histamine, tyramine and glutamate did not result in the activation of SmGAR (Figure 16A).94 The effect of acetylcholine was dose-dependent (Figure 16A).94 Although the receptor could be activated by the cholinergic agonist carbachol, the response was lower than that observed with acetylcholine (Figure 16C).94 The results showed that SmGAR forms a constitutively active receptor that is selectively activated by acetylcholine. Section 6.2 discusses the pharmacological profile of SmGAR.

5.2.2 Non-conservative amino acid substitutions may contribute to SmGAR’s constitutive activity To assess whether SmGAR contains unique structural features that can serve as an explanation for its high basal activity, the receptor was compared to other cholinergic receptors.94 For this purpose, a structural alignment of GAR-like receptors and mAChRs from nematodes, humans and various Platyhelminthes was generated. Furthermore, the crystal structure of the rat M3 muscarinic receptor (4daj) was utilized as a template to generate a homology model of SmGAR. The results showed that SmGAR contains typical hepta-helical architecture of class A GPCRs. Moreover, the transmembrane regions closely align with the template (Figure 17A).94 Various amino acids that are implicated in ligand binding were identified.102 Firstly, three lid-forming tyrosines of the 7.39 6.51 3.33 binding pocket of the receptor (SmGAR Tyr898 , Tyr869 , Tyr231 ) were identified.

Secondly, SmGAR Asp2303.32 is the highly conserved TM3 aspartate and is important for the binding of the positively charged headgroup of acetylcholine and related ligands. However, amino acids substitutions that could possibly affect signaling activity or/and binding affinity were also found. To illustrate, substitutions were observed in the cytoplasmic end of TM6 and the TM3/il2 interface (Figure 17B). Both regions are known to be important in the conformational activation of GPCRs and the coupling of G proteins.103 Furthermore, the TM6 asparagine of mammalian muscarinic receptors (Asn6.52), which is known to play an important role in hydrogen bonding within the binding pocket of the ligand, is replaced with histidine (His8706.52).

Previous research has established that there are regions that are important for constitutive activity of receptors. These regions can affect intra- and inter-helical bonding interactions, which can result in a conformational shift between active and inactive states.94 One of these regions is known as the ‘ionic lock’ of rhodopsin-like receptors. In this region, an electrostatic interaction is formed between glutamate of TM6 (Glu6.30) and arginine of TM3 (Arg3.50), which keeps together the cytosolic ends of the helices. As a result, the receptor is stabilized in an inactive conformation. When this interaction is disrupted, it can result in a constitutively active GPCR.104 Structural analysis of SmGAR showed that the arginine of TM3 is conserved (Arg2483.50) (Figure 17B).94 However, the receptor has an alanine (Ala8486.30) instead of the critical glutamate of TM6, and a phenylalanine is replaced by cysteine (Cys2503.52) near the interacting site of TM3. These results are likely to be related to the constitutive activity of SmGAR. Sequence analysis of invertebrate GAR-like sequences showed that most carried the TM6 Glu → Ala substitution (Figure 17B).94 This may suggest a family of invertebrate GARs that are constitutively active or it points towards a difference in the mechanism of conformational activation between vertebrate and invertebrate GPCRs.

Taken together, these results suggest that there is an association between the non-conservative amino acid substitutions in the ‘ionic lock’ region of SmGAR and the receptor’s constitutive activity. The next section investigates the role of SmGAR in S. mansoni.

Figure 17: Non-conservative amino acid substitutions may play a role in SmGAR’s constitutive activity. A) The crystal structure of the rat M3 muscarinic receptor (4daj, blue was utilized as a template to generate a homology model of SmGAR (red). ‘Ionic lock’ residues of members of class A GPCRs are visualized at the cytosolic ends of TM3 and TM6. B) Structural alignment of invertebrate and vertebrate muscarinic receptors. A part of TM3 and TM6 is shown. Mammalian muscarinic receptors (M1–M5 and invertebrate receptors (GAR or mAChR) are used for the alignment.94 5.2.3 SmGAR is involved in S. mansoni larval motility SmGAR is suggested to be upregulated in early larval stages and acetylcholine is known to be involved in controlling worm movement.37 To investigate whether SmGAR is involved in larval motility, an RNAi phenotypic assay was utilized.94 Schistosomula that were newly transformed were treated with a pool of heterogeneous siRNA specific to SmGAR. Subsequently, the effect on the motility of the parasites was measured using a quantitative imaging assay. The control group consisted of schistosomula treated with nonsense scrambled siRNA. Analysis of the results showed that treatment with SmGAR siRNAs resulted in a significant (P < 0.01) reduction (±70%) in the frequency of body movement in comparison to the control group (Figure 18A).94 These results are in agreement with the findings that were determined by qRT-PCR, which showed an almost complete knockdown of the transcript (Figure 18B).94 In contrast, the expression of an unrelated gene (SmACC-1) did not change, which suggest that the suppression of the receptor was specific. Overall, these results indicate that SmGAR plays a role in larval motility and its activity results in the movement of young larvae. This suggests a potential role for SmGAR in early parasite migration. This transcript levels of SmGAR showed a bias towards sM, bM, and sF (sM-bM-sF group; Figure 8).17 A role for SmGAR in locomotion could be possible because this process has similar importance in sM, bM, and sF.

Figure 18: The motor behavior of S. mansoni is affected by silencing of acetylcholine receptor SmGAR. Newly transformed schistosomula were treated with siRNA specific to SmGAR or irrelevant siRNA (50 nM). 24 hours post-transfection the animals were tested for motor phenotype or for measuring of mRNA expression levels of SmGAR. A) 70% reduction of frequency of body movements was observed in the schistosomula that were treated with SmGAR siRNA compared to the negative control. B) RNA that was collected from treated animals was oligo-dT reverse-transcribed. Subsequently, the obtained cDNA was utilized as a template for qPCR. An off-target (irrelevant) gene was used as a control and a housekeeping gene was used to normalize the data. Expression of the receptor and control were calculated as percentage of remaining expression in the samples that were treated with SmGAR siRNA relative to those that were treated with irrelevant siRNA, by means of the Pfaffl’s method.94

Since acetylcholine is known to inhibit the movement of schistosomes, the stimulating function of SmGAR seems contradictory. This may be explained by the fact that there are a lot of cholinergic receptors in schistosomes, of which some do have inhibitory effects on motility.30 Stimulation of several of these receptors at the same time may result in the observed flaccid paralysis caused by acetylcholine.95 Alternatively, SmGAR may be involved in a negative feedback mechanism. Loss of receptor activity would then result in increased acetylcholine signaling and reduced larvae motility. A similar mechanism has been described for C. elegans receptor GAR-2.105

To conclude, SmGAR is the first example of a cholinergic GPCR that stimulates motility in S. mansoni larvae. The receptor is constitutively active but can be activated above it elevated baseline by acetylcholine. Due to the importance of motility in the life cycle of S. mansoni, SmGAR is a promising antischistosomal drug target. Moreover, the currently available drug to treat Schistosomiasis, PZQ, is not effective against juvenile schistosomes.8 Drugs that target SmGAR are likely to be effective against larvae since the receptor is upregulated in that life stage. Future research should focus on the mode of action of SmGAR. Section 6.2 discusses the pharmacological profile of SmGAR. The next section analyzes the characterization of dopamine receptor SmD2.

5.3 Dopamine receptor SmD2 The ability to migrate in the bloodstream of the host is crucial for the survival of S. mansoni (Chapter 1). This migration is coordinated by the neuromuscular system of S. mansoni. The controlled movement is enabled by the subtegumental nerve plexuses that innervate the body wall musculature that coordinate the activity of various types of fibers.106 Receptors that play a role in the motility of the parasite have strong potential for antischistosomal drug targeting. Previous studies have shown that when S. mansoni is treated with dopamine, relaxation of the body wall muscles occurs.106 More specifically, relaxation of the longitudinal as well as the circular muscle was observed. Furthermore, this effect could be reversed by addition of a dopamine antagonist (spiroperidol), which suggest that this effect was receptor-mediated. However, there is little published data on the mechanism of this inhibition. This section discusses the functional characterization and immunolocalization of a dopamine receptor, SmD2, in S. mansoni.

5.3.1 SmD2 is selectively activated by dopamine when expressed in yeast The putative type 2 dopamine GPCR was selected from the S. mansoni database based on the observation that it has sequence homology with other type 2 dopamine receptors.66 The receptor shares sequence homology with D. melanogaster (52%) and A. mellifera (51%). The level of homology with human D2 is lower (43%). The full length SmD2 was cloned using RT-PCR. The receptor has seven predicted transmembrane domains and three serine residues in TM5 (S5.42, S5.43 and S5.46) that undergo interactions with the hydroxyl groups on the catechol ring.107 Furthermore, it has a highly conserved aspartate in TM3 as well as multiple motifs that characterize family A GPCRs.108 These conserved residues indicate that SmD2 most likely is a functional dopamine receptor.

To test whether SmD2 is a functional receptor, the full-length cDNA was expressed in yeast (S. cerevisiae). The strain that was used (CY13399) expresses a HIS3 reporter gene under the control of a FUS1 promoter.109 It also contains an integrated copy of a chimeric Gα gene in which the last five codons were of human Gαs instead of those of native yeast.110 Strains carrying Gαi2, Gα12, Gαo and Gαq yielded no receptor activity. Receptor-induced cell growth was measured to quantify receptor activity using a fluorometric Alamar Blue assay. The assays were performed in media that lacks histidine. The cells were treated with various amines (10-4 M; dopamine, histamine,

48 octopamine, epinephrine, serotonin, tyramine). The results revealed that SmD2 is selectively activated by dopamine (Figure 19A).66 Remarkably, SmD2 was constitutively active in absence of agonist. This propensity towards spontaneous activation has been described for other GPCRs.111 For example, the schistosome receptor SmGAR exhibits constitutive activity when expressed in yeast (section 5.2).94 The functional assay was repeated with different concentrations of dopamine. These results showed that the dopamine stimulates the receptor in a dose-dependent manner (Figure 19B).66

Figure 19: SmD2 is expressed in yeast and responds selectively to dopamine. A) SmD2 cDNA was expressed in S. cerevisiae strain CY13399 and grown in a histidine-deficient medium. Mock-transfected controls were used to normalize the data. The cells were treated with a test agonist (10-4 M) or vehicle (-). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth (fluorescence). SmD2 exhibited constitutive activity, but was further activated by addition of dopamine (DA) (P < 0.05). No significant effect was measured in the cells that were treated with other test agonists. (epinephrine, EPIN; histamine, HIST; octopamine, OCT; serotonin, SER; tyramine, TYR). B) Experiments were repeated using various concentrations of dopamine.66

5.3.2 SmD2 signals through stimulation of cAMP The majority of the studies that express cloned S. mansoni cDNA used yeast as an heterologous expression system. The reason for this is that problems can arise when schistosome GPCR is attempted to be expressed in mammalian cells.79 The expression of S. mansoni cDNA in HEK293 cells can be increased by the usage of codon optimization.112 A part of the S. mansoni cDNA is rewritten according to human preferred codon usage. To investigate the signaling mechanism of SmD2, the cDNA was expressed in HEK293 cells.66 Subsequently, the effect of receptor activation

49 on Ca2+ and cAMP levels was measured. The expression of SmD2 in HEK293 cells was confirmed by in situ immunofluorescence (Figure 20A).66 The transfected HEK293 cells were treated with different concentrations of dopamine and were subsequently tested for cAMP using a competitive immunoassay. D2 dopamine receptors in other organisms have been described to inhibit adenylate cyclase, which result in decreased cytosolic cAMP levels. In order to test whether the same effect can be observed for SmD2, the transfected cells were treated with forskolin and dopamine. However, no decrease in forskolin-induced cAMP levels was observed. In contrast, a stimulation of cAMP was observed in the transfected HEK293 cells (Figure 20B).66 Dopamine was shown to selectively stimulate cAMP in a dose-dependent manner (EC50 =0.17±0.31 µM). No response was measured when intracellular calcium levels were measured after receptor activation with dopamine.66,113,114 This means that SmD2 does not signal through the Ca2+/phosphoinositol pathway.

These results showed that SmD2, which exhibits sequence homology with other D2-like receptors, is most likely Gs-coupled and signals through stimulation of cAMP levels system. This is different compared to mammalian D2 dopaminergic receptors, since they signal by stimulating the Ca2+/phosphoinositol pathway or by decreasing cAMP levels.115

Figure 20: SmD2 is expressed in HEK293 cells and signals through increased cAMP levels. A) SmD2-pCI-neo construct was expressed in HEK293 cells. The cells were treated with an anti-SmD2 antibody, after which FITC labeled secondary antibodies were added. Confocal microscopy was used to visualize the cells. SmD2 immunoreactivity (green fluorescence) was detected in the cells, while no immunoreactivity was visible in the controls. The nuclei are stained blue by DAPI. B)Various dopamine (DA) concentrations were used to treat HEK293 cells that expressed SmD2 or empty plasmid (mock). Subsequently, the cells were lysed and tested for cAMP by the means of a competitive immunoassay.66

5.3.3 SmD2 is expressed in the somatic muscles of S. mansoni Section 3.2 discussed that SmD2 (Smp_127310) is expressed in non-gonad tissue of both female and male S. mansoni.17 SmD2 grouped in the ‘whole worm’ group, meaning that there is a balanced transcription rate among both male and female S. mansoni independently of pairing (Figure 8).17 To shed light on the mechanism of SmD2, the tissue distribution was determined in different life- stage of S. mansoni using an anti-SmD2 antibody and a FITC-conjugated secondary antibody.66 Furthermore, muscle fibers were visualized by co-labelling the animals with TRITC-phalloidin.

The results revealed that SmD2 is expressed in the subtegumental region independent of life-stage. It is predominantly expressed in the acetabulum and the subtegumental somatic musculature of all larval stages.66 In adult schistosomes, the receptor was mainly present in the somatic muscles and in a lower amount in the muscular lining of the caecum. The results of the experiments performed on the animals that were stained with phalloidin revealed significant co-localization of phalloidin and SmD2 immunofluorescence in somatic muscle fibers. Co-localization was detected in the circular as well as the longitudinal fibers of the body wall. The authors report that SmD2 appears to be upregulated in schistosomula and cercariae compared to adult worms. However, this is not confirmed at mRNA level yet. This upregulation has been previously described for other S. mansoni GPCRs (Chapter 4). For example, SmGPR-1 is particularly upregulated during the development of early sporocyst and schistosomula.77

These results begin to unravel the mechanism of SmD2 in S. mansoni. Since no expression was detected in the main nerve cords of the central nervous system or the cerebral ganglia, SmD2 most likely does not signal through centrally located neurons. Co-localization with somatic muscle fibers indicates that SmD2 is found on the myofiber membrane or other neuronal elements. The somatic muscle of flatworms is generally of a smooth type.29 In other organisms, increased cAMP levels lead to inhibition and thus relaxation of smooth muscle. cAMP blocks the activation of myosin light chain kinase (MLCK), which mediates the interaction between myosin and actin in contractile systems.116 Since SmD2 is coupled to the stimulation of adenylate cyclase, dopamine could cause inhibition of muscle contraction by activating receptors that are located on the muscle membrane, and thereby increasing cAMP levels within the myofibers. Another potential mechanism of SmD2 in S. mansoni involves modulation of the activity of other transmitter receptors that are present on

51 the musculature or modulation of transmitter release. SmD2 is not the only receptor in S. mansoni that is activated by dopamine. SmGPR-3 is also activated by dopamine and is suggested to mediate an indirect neuronal pathway (section 5.4.8).80 This indicates that there that there are multiple dopaminergic mechanisms that control worm motility.

In conclusion, SmD2 is a D2-like receptor in S. mansoni that is selectively activated by dopamine. The receptor signals through an atypical pathway. SmD2 is expressed in the somatic muscles, which suggest that it plays a role in the previously shown effects of dopamine on worm motility. This indicates that SmD2 could be a potential antischistosomal drug target. Section 6.3 discusses the pharmacological profile of SmD2. The next section discusses the characterization of receptors that are a member of a novel clade of schistosome-specific receptors.

5.4 A novel clade of schistosome-specific, SmGPR-like receptors 5.4.1 Histamine signaling in S. mansoni host–parasite interactions As described in section 1.1, the life cycle of S. mansoni is complex and requires a freshwater snail as an intermediate host. The well-developed nervous system of S. mansoni is necessary to coordinate the various life stages.117 In section 5.2, the effect of neuroactive compound acetylcholine on SmGAR was discussed. However, multiple molecular mechanisms are involved in neuronal signaling.30 The neurotransmitters that are involved in these systems include the biogenic amines (BAs). Extensive research has shown that BAs are widely distributed in the nervous system of schistosomes and they interact with a variety of receptors.28 BA signaling has been shown to be involved in the control of neuromuscular function, motility and larval development.118 Although research has been carried out on the endogenous BA system of S. mansoni, there is still very little scientific understanding of the role of BAs in the interaction between schistosomes and the host.

Since schistosomes respond to exogenously applied BAs in vitro, it is plausible that the parasites respond to BAs that are present in the host. This could occur via activation of BA receptors located on sensory nerve endings of the parasite surface or through uptake of host BAs via surface transporters (Figure 21). Two receptors have been described that are located on sensory nerve endings: SmGPR-1 and SmGPR-2.76,77 Studies on adult schistosomes and schistosomula have

52 revealed that SmGPR-1 is enriched on the surface of the parasite.77 It is possible that this receptor responds to exogenous histamine. Both receptors respond selectively to histamine and are class A GPCRs (section 5.4.3 & 5.4.6).

Figure 21: Two mechanism by which histamine of host origin could interact with S. mansoni.117

Although some BAs are endogenously synthesized in schistosomes,28,119 they take up exogenous amines through the tegument as well.120 Studies have found many histamine-containing neurons in S. mansoni.76 However, it is unclear whether histamine is entirely of host origin or also endogenously synthesized in the parasite. Computational studies have not been able to find a homolog of histidine decarboxylase, which is the enzyme that catalyzes the decarboxylation of histidine into histamine.117 However, another study states that a BLAST analysis of the parasite’s genome database found a potential orthologue of the same enzyme.77 It remains unclear whether S. mansoni is able to synthesize histamine endogenously. In contrast, previous studies have characterized a functional tryptophan hydroxylase as well as a tyrosine hydroxylase in S. mansoni, suggesting that schistosomes are capable of synthesizing serotonin and catecholamines endogenously.119,121 It may be the case that histamine is rapidly degraded after its release or it synthesis rate is relatively low. In agreement with this hypothesis, histaminase activity in S. mansoni has been described.122 A high histamine degradation rate could also explain the low levels of histamine present in S. mansoni tissue.

Histamine was localized in adult S. mansoni by a commercially available monoclonal anti-HA antibody.76 The results indicated widespread and abundant HA immunoreactivity in the peripheral nervous system of S. mansoni. Histaminergic processes and cell bodies are present in the innervation of the suckers, both oral and ventral, as well as in the nerve plexus that supplies the body wall musculature. No significant HA labeling was identified in the major nerve cords of the central nervous system or the brain region. These results indicate that histamine is likely to act as a modulator or transmitter of the peripheral nervous system rather than the central nervous system. The widespread and abundant distribution of histaminergic neurons indicates that histamine is an important neuroactive system in S. mansoni.

The precise biological role of histamine in flatworms remains unknown. However, research suggest that it affect the musculature in a dose-dependent manner. Histamine was shown to stimulate motility in S. mansoni.123 Furthermore, the administration of promethazine, an antihistamine, to schistosomes resulted in paralysis, which could be reversed by histamine. This demonstrates that importance of histamine receptors in S. mansoni as well as the potential of these receptors for drug targeting. SmGPR-1 and SmGPR-2 are both located on sensory nerve endings, and could be involved in histamine signaling in host-parasite interactions. SmGPR-1 and SmGPR-2 were found to have a similar developmental expression patterns (section 4.2).76,77 The upregulation of SmGPR- 1 in parasitic stages in comparison to free-swimming stages suggest that this receptor has a fundamental role in the establishment and/or maintenance of parasitism within the hosts. Since their developmental patterns are similar, it is suggested that these kind of receptors are especially important during the development of early schistosomula. The following sections describe the functional analysis and characterization of SmGPR-1, SmGPR-2 and SmGPR-3. Furthermore, the tissue distribution of these receptors is analyzed. The next section describes the clade to which these receptors belong (SmGPR-like clade).

5.4.2 SmGPR-1, SmGPR-2 and SmGPR-3 belong to a new clade of amine-like receptors The first invertebrate histamine receptor that was described is SmGPR-1 (Smp_043260, SmGPCR).79 This receptor shares about the same level of sequence homology with all types of BA GPCRs (± 30%), but it does not have direct orthologue. However, SmGPR-1 shares a long third intracellular loop with the H1 type. In addition, it shares various conserved substitutions in TM4

(Ala155), TM5 (Arg189/Lys) and in the second intracellular loop (Arg143, Arg145/Lys, Ala148) with the H1 type. However, there is a remarkable differences between histamine receptors and SmGPR- 1. For example, histamine receptors have a charged (Glu) or polar (Thr/Asn) residue in TM5, while SmGPR-1 has a glycine (Gly196). The charged/polar residue in TM5 has been implicated in anchoring the imidazole nitrogen of histamine.

Bioinformatics analysis of the genome of S. mansoni revealed that 16 full-length GPCRs showed significant homology with aminergic receptors from vertebrates and invertebrates. A new clade of amine-like GPCRs was found that included six of these sequences that are structurally related to SmGPR-1. A second member of this clade is SmGPR-2 (Smp_043340) (43.0% homology).76 The most closely related sequences to SmGPR-2 are predicted S. japonicum and S. mansoni receptors. Furthermore, SmGPR-2 shows homology with BA GPCRs from other organisms, such as mammals (i.e. H1R, H2R, H3R, and H4R), planarians and insects. However, the level of homology is generally lower (≈30%). The third member, SmGPR-3, is most closely related to SmGPR-2 (46.9% homology), SmGPR-1 (53.4%), Smp_043270 (45.5%) and FN328430 (S. japonicum, 46.1%). Furthermore, this receptor is related to BA receptors from other organisms as well as to schistosome BA receptors.

Members of this novel SmGPR-like clade, including SmGPR-1, SmGPR-2 and SmGPR-3, have the typical heptahelical topology and all of the characteristic motifs of class A GPCRs. These include the NPxxY motif of TM7 and a DRY (Asp-Arg-Tyr) motif at the intracellular boundary of TM3. Furthermore, the aromatic cluster FxxCWxPFF of TM6 was identified, which has been implicated in the activation of the receptor and binding of biogenic amines (BA).124–127 The aromatic residues would normally be expected to contribute to the binding of the catechol moiety 80 (for example in dopamine) or other aromatic moieties. For example, in serotonin receptor 5-HT2A the charged terminal amine group is anchored by Asp-155 to facilitate interactions of the aromatic moiety of indoles with aromatic residues in TM6, which leads to receptor activation.125

Remarkably, the majority of SmGPR-like receptors do not have a functional aspartate (D3.32) of TM3 (Figure 22),76 which is a conserved residue in all known BA GPCRs, and is thought to be fundamental for receptor activity.126,128 The other predicted S. mansoni BA receptors also contain

D3.32, whereas most SmGPR-like receptors carry an asparagine at this position. The only exception to this is SmGPR-3 (Smp_043290), which does have D3.32 (Figure 22).76 Modelling studies suggested that this particular aspartate functions as an anchoring point for various amines.76,129 More specifically, the carboxylate on the side chain of D3.32 is thought to form a salt bridge with the protonated amino moiety of the various biogenic amines.128,130 Point mutations D3.32A and D3.32N resulted in a lower or loss of binding affinity in various receptors, such as histamine H1 and H4 GPCRs.131–133 Therefore, the lack of D3.32 in the majority of SmGPR-like receptors suggest that the binding pocket may be fundamentally different. An unique glutamate residue (E3.20) is present in in every SmGPR-like receptor (Figure 22).76 This residue is not present in other aminergic receptors. Possibly, this substitution is specific for schistosomes and compensates for the lack of D3.32. It is suggested that the asparagine substitution happened after the separation of parasitic and free-living flatworms and is specific to parasites.

In summary, a new clade constitutes of amine-like S. mansoni GPR receptors (SmGPR). The SmGPR homologues are characterized by the substitution of the highly conserved aspartate D3.32 of TM3 to an asparagine. The only exception to this is SmGPR-3 (Smp_043290). The next section describes the functional analysis of SmGPR-1.

Figure 22: SmGPR-like sequences (horizontal box) do not have the conserved aspartate D3.32 of TM3 (right vertical box). A ClustalW alignment was performed. TM domains II and III are shown and invariant residues are marked by asterisks. SmGPR-like receptors have an unique glutamate (E3.20) (left vertical box). Shaded areas indicate regions of conserved or identical sequences.76

5.4.3 SmGPR-1 is selectively activated by histamine and is a Gq/11-coupled receptor SmGPR-1 is the first invertebrate histamine receptor that was described. Like most receptors that are part of the SmGPR clade, SmGPR-1 does not have the aforementioned conserved aspartate (D3.32) of TM3. In this section, the characterization of SmGPR-1 is described. Furthermore, the significance of the D3.32 substitution is analyzed. A standard RT-PCR reaction was used to amplify the full-length SmGPR-1.79 It was codon-optimized for expression in transfected COS7 and HEK293 cells. PCR was performed to insert a FLAG epitope,134 a Kozak sequence and a C- terminal oligohexahistidine tag.135 These were added to monitor the receptor expression by in situ immunofluorescence. In addition, a stop codon followed by a single adenosine was added. This increases the efficiency of translation termination in the transfected cells. Similarly, the Kozak sequence increases the efficiency of translation initiation. The results of the in situ immunofluorescence showed that SmGPR-1 is targeted to the cell surface and can be transiently expressed in COS7 as well as HEK293 cells.

Various amine ligands were tested on SmGPR-1 that was transiently expressed in COS7 or HEK293 cells. cAMP signaling was measured using 293CRESEAP cells that were transfected with SmGPR-1.136 Ligands that were tested included octopamine, histamine, tyramine, adrenaline, acetylcholine, serotonin, noradrenaline and dopamine. All ligands were tested in the absence or presence of forskolin. The results showed that no significant response could be measured compared to the controls. However, positive results were obtained using an aqueorin assay, which is a sensitive method for calcium measurement.137 SmGPR-1 and an aqueorin expression plasmid were transfected in COS7 cells. The results showed that SmGPR-1 is responsive to histamine dose- 79 dependently (EC50= 0.54 ± 0.05 µM) (Figure 23). The other ligands that were tested did not have any observable effect. When a mutant form of SmGPR-1 (SmGPR-1.Asp111) was used, in which Asn111 (Asn3.32) of TM3 was mutagenized to an aspartate, no significant difference was observed in the dose-response curve (Figure 23).79 This is finding was unexpected and suggests that Asn111 is not directly involved in the activation of SmGPR-1. This is surprising because in mammalian H1 receptors, this aspartate of TM3 is highly conserved and believed to be important for the binding of amines.138 Even without this conserved residue, SMGPR-1 is activated by histamine, which points towards a different conformation of the binding pocket compared to other BA GPCRs.

Figure 23: SmGPR-1 is activated by histamine. Dose-dependent activation of SmGPR-1 (solid squares) expressed in COS7 cells cotransfected with an aqueorin expression plasmid. Replacement of SmGPR-1 with SmGPR1.Asp111 (open circles) did not result in a change in the dose-response curve.79

These results indicate that histamine is a selective agonist of SmGPR-1. Furthermore, the results suggest that SmGPR-1 is a Gq/11-coupled receptor and transduces through the inositol phosphate 79 pathway. The EC50 for histamine was found to be in the micromolar range. The EC50 for histamine indicates low-affinity ligand binding. Remarkably, most invertebrate amine GPCRs that have been tested in (mammalian) heterologous expression systems exhibit micromolar affinities for their ligands. It is unclear whether this is an intrinsic property of the receptors or an artefact caused by the heterologous expression systems. It is important to take into consideration that SmGPR-1 may be activated in vivo by a structurally similar to histamine, unknown amine in S. mansoni.

A previous study showed that exogenous histamine increased the motility of S. mansoni worms in vitro,123 which suggest that histamine possibly functions as a positive modulator of neuromuscular activity in S. mansoni.41 Currently, it is unclear if the receptor that causes this effect is involved in endogenous histamine signaling or if it plays an unknown role related to exogenous histamine (section 5.4.1). Due to host’s immune response, S. mansoni comes in contact with high levels of blood histamine. For this reason, it is possible to hypothesize that a S. mansoni histamine GPCR is directed towards host-derived histamine. This receptor could be involved in a mechanism that evades the immune response of the host.

In summary, SmGPR-1 selectively responds to histamine when expressed in mammalian cells. Activation by histamine resulted in a dose-dependent rise in intracellular calcium, which suggest that SmGPR-1 is coupled to Gq/11 proteins and the inositol phosphate signaling pathway. The next section describes the structural analysis of SmGPR-1 and its tissue distribution.

5.4.4 SmGPR-1 exists as a monomer and dimer in transfected HEK293 cells and schistosomes To further characterize SmGPR-1, polyclonal antibodies were raised to the recombinant third intracellular loop. An ELISA assay, Western blotting studies and in situ immunofluorescence revealed that the antibody could recognize the complete protein in transfected HEK293E cells as well as in S. mansoni extracts. Immunoprecipitation of SmGPR-1 from schistosome extracts confirmed that SmGPR-1 exist as a monomer (65 kDA) as well as a dimer (130 kDa). The dimer showed resistance towards reducing and denaturing agents. Remarkably, when the pH was gradually increased from 3 to 7.4, the 130 kDa band appeared while the 65 kDa band disappeared. This indicates that the dimer and monomer are interconvertible and the dimer involves non- covalent interactions. The higher molecular weight forms could be an artifact of the solubilization conditions. Recent studies, however, have shown that GPCR oligomerization is an important part of the function and structure of the receptors.139 Moreover, dimerization plays an important role in pharmacology, signaling and receptor trafficking. The existing body of research on dimers suggests that they involve non-covalent interactions and/or intermolecular disulfide linkages.140,141 The observation of the SmGPR-1 dimer in preparations of recombinant as well as native receptors indicates that this GPCR dimer is biologically relevant.

5.4.5 SmGPR-1 potentially has functions in the tegument and muscle To investigate tissue distribution of SmGPR-1 in S. mansoni, an anti-SmGPR-1 antibody was used.77 Adult worms, schistosomula, sporocysts and miracidia were probed with the polyclonal antibody, after which a FITC-labeled secondary antibody was added. In addition, muscles and cytoskeletal elements were labeled by treating the various S. mansoni life-stages with rhodamine- conjugated phalloidin. 142–144 The results showed that no SmGPR-1 immunoreactivity was found in miracidia. In contrast, immunoreactivity was found in sporocysts, particularly localized in cells within the parenchymal matrix and the surface tegument (Figure 24A-B).77 No immunoreactivity was found in the controls (Figure 24C-D). Schistosomula exhibited widespread green fluorescence

60 localized in the acetabulum, parenchyma, anterior muscle cone, tegument, and within the esophagus (Figure 24E-G).77 The experiments performed on the animals that were stained with phalloidin revealed significant co-localization of phalloidin and SmGPR-1 immunofluorescence in the subtegumental musculature. The results suggested that the receptor co-localizes with muscle, especially in the outer layer beneath the tegument (Figure 24I-K).77 In adult worms, immunoreactivity was found in the tubercles of the male tegument (Figure 25A, C, D), whereas no activity was found in the controls (Figure 25B).77 Co-localization of the receptor with phalloidin revealed that SmGPR-1 is expressed in the subtegumental musculature, especially in the head region and the anterior end (Figure 25E).77

Figure 24: Localization of SmGPR-1 in S. mansoni larval stages. Sporocysts and schistosomula were treated with anti-SmGPR- 1 antibodies or controls, after which FITC labeled secondary antibodies were added. Sporocysts showed immunoreactivity (green) on the tegument (arrows) and within the parenchyma (A, B), while no immunoreactivity was visible in the controls (C, D). Muscle and cytoskeletal elements were labeled with (TRITC)-labeled phalloidin (red). This did not show apparent co-localization (B). Probing of schistosomula 4 days (E) or 8 days (G) with anti-SmGPR-1 showed immunoreactivity in the tegumental region, parenchyma cells, acetabulum and the esophagus. Immunoreactivity could also be detected in the posterior end of 8-day schistomula, particularly in the acetabulum (H). No fluorescence was detected in the controls (F). Schistosomula (28-day) were treated with anti-SmGPR-1 antibodies and phalloidin. This produced red fluorescence in the circular, major longitudinal and oblique body wall muscles (I). Co-localization was detected (yellow) in the musculature of the acetabulum (arrow), the subtegumental musculature (box), and in the muscle cone of the head region (arrow) (I, J). Co-localization was also detected in the outer layer of the musculature (K).77

Figure 25: Localization of SmGPR-1 in adult S. mansoni. Male worms were treated with anti-SmGPR-1 antibodies or controls, after which FITC labeled secondary antibodies were added. SmGPR-1 immunofluorescence (green) was detected in the tubercles of the dorsal tegument (A, C), while the control showed only background fluorescence (B). Muscle and cytoskeletal elements were labeled with (TRITC)-labeled phalloidin (red). This produced a pattern of green fluorescent tubercles surrounded by red tegumental spines (D). Co-localization of phalloidin and SmGPR-1 was found in in the subtegumental musculature (yellow, E).77

The results showed that SmGPR-1 fluorescence staining was predominantly found in the parasitic stages compared to the free-swimming miracidia. This result is in agreement with gene expression data (section 4.2).77 This further supports the hypothesis that this receptor may have a fundamental role in the establishment and/or maintenance of parasitism within the hosts. However, it is not yet clear what this role is. The results revealed that SmGPR-1 is expressed in the body wall musculature, which suggest that it is involved in motility. In schistosomula, SmGPR-1 expression was found in the musculature of the suckers. This suggest that the receptor may play a role in neuromuscular transmission or modulation. Remarkably, no expression of SmGPR-1 was detected in the central nervous system. SmGPR-1 expression was detected in the tegument in all parasitic S. mansoni life-stages. The observation that SmGPR-1 is localized on the surface suggest that they are likely to be activated by host-derived signals and involved in host-parasite interactions (section 5.4.1). Evidence suggest that parasites increase vascular permeability by using the host’s histamine system.81–83 This facilitates passage through blood vessels during larval migration. It may be the case that the upregulation of S. mansoni’s own histamine system is involved in this. A tegumental

62 histamine GPCR may be involved in a system that responds to changes in exogenous histamine concentration. This response may entail an increase in motility. SmGPR-1 expression was also detected in the tubercles, which are present in sensory nerve endings and ultimately connect to the central nervous system.145 This suggest that activation of this GPCR could lead to signaling through chemosensory circuits, thereby influencing the behavior of the worm.

It is hypothesized that there are two histamine systems operating in S. mansoni.77 The first one being endogenous and controlling mainly the musculature. The second system could be exogenous and located on the tegument, and is likely to be activated by host-derived histamine or histamine- like molecules. In sporocysts, the SmGPR-1 is primarily expressed in the tegument and its surface. At this stage, no association with muscle fibers could be detected. The receptor is more widely expressed in schistosomula and adult schistosomes. In these stages, SmGPR-1 was detected in various tissues, the tegument and in the musculature. These results further support the idea of a two histamine signaling systems in S. mansoni. As mentioned earlier (section 4.2), SmGPR-1 was found to be significantly upregulated at the RNA levels in parasitic stages in comparison to free- swimming stages.77 The most prominent upregulation was detected in sporocysts. These observations suggest that SmGPR-1 has a fundamental role in the establishment and/or maintenance of parasitism within the hosts.

Overall, these results indicate that SmGPR-1 is an important histamine receptor in S. mansoni that potentially plays a role in the tegument and the control of muscle function. Future research should focus on determining whether SmGPR-1 is the receptor that causes the previously described effects of histamine on worm motility.123 This could possibly be achieved by silencing SmGPR-1 expression by RNAi in S. mansoni. The next section discusses the functional analysis and immunolocalization of SmGPR2, which is a receptor that is a member of the same clade as SmGPR-1.

5.4.6 SmGPR-2 is constitutively active and selectively activated by histamine Functional expression studies of SmGPR-2 were conducted by expressing the cDNA in yeast (Saccharomyces cerevisiae).76 A histidine auxotrophic strain (CY13399) was used that expresses a HIS3 reporter gene under the control of a FUS1 promoter.109 It also contains an integrated copy of

63 a chimeric Gα gene in which the first 31 and last five codons were of human Gαq instead of those of native yeast.110 Strains containing Gαi2, Gα12, Gαo and Gαs yielded lower or no receptor activity. The activation of the recombinant GPCR was quantified by measuring yeast growth in a histamine-deficient medium, using a fluorometric Alamar Blue assay. The cells were treated with common biogenic amines (adrenaline, dopamine, histamine, noradrenaline, octopamine, serotonin, tyramine). The results revealed that the receptor was constitutively active and selectively further activated by histamine (P < 0.01) (Figure 26A).76 A dose-dependent response was found when increasing concentrations of histamine were used (Figure 26B).76 Remarkably, the half-maximum effective concentration (EC50) was in the micromolar range. The authors reported that in mammalian histamine receptors, this value is usually lower. However, the EC50 is comparable to 146 that of the mammalian H1/H2 class. In SmGPR-1 expressing cells, the EC50 for histamine was also in micromolar range. The authors of this study reported that this value is comparable to the mammalian H2 class.79 Nonetheless, it could be that D3.32N substitution (section 5.4.2) lowers the binding affinity. Similarly, the high basal activity of the receptor could also be due to the D3.32N substitution. Previous studies showed that single-point mutations of D3.32 in GPCRs can lead to 147,148 higher agonist-independent activity levels. However, both the high EC50 value and the high basal activity could be a result of heterologous expression in yeast.

Figure 26: Functional expression studies of SmGPR-2 in yeast. A) SmGPR-2 cDNA or empty plasmid (mock) was expressed in S. cerevisiae strain YEX108 and grown in a histidine-deficient medium. The cells were treated with one of the biogenic amines (10- 4 M) or vehicle (ND). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth (RFU, relative fluorescence units). SmGPR-2 exhibited constitutive activity, but was further activated by addition of histamine (HA). No activity was measured in the cells that were treated with other biogenic amines. (adrenaline, A; dopamine, DA; noradrenaline, NA; octopamine, OA; serotonin, 5-HT; tyramine, TA). B) Experiments were repeated using various concentrations of histamine.76

The constitutive activity of SmGPR-2 could be relevant in vivo. Various mammalian receptors have been shown to be constitutively active in vivo, e.g. 5HT4, histamine H3 receptor, PTH receptor and b1-adrenoceptor.149 In some cases, spontaneous activity of these receptors have been linked to disease. Furthermore, GPCRs encoded by herpesviruses are constitutively active, and this is believed to contribute to the mechanism of infection.150,151 Constitutive activity of GPCRs has been linked to single nucleotide polymorphisms, RNA editing and/or splicing events that cause disruption of the normal constraints on the activation of GPCRs.152 The amino acids that are involved in these constraints are D3.49, R3.50, E6.30 and T6.34. Many of these are conserved in SmGPR- 2. However, the absence of D3.32 in SmGPR-2 might contribute to the destabilization of the inactive conformation. Remarkably, another receptor that was found to have high basal activity is SmGAR (section 5.2). This receptor has a conserved R3.50 but the receptor has an alanine (Ala8486.30) instead of the critical glutamate of TM6.

Overall, these results indicate that SmGPR-2 is a constitutively active receptor that is selectively activated by histamine. The next section examines the tissue localization of SmGPR-2.

5.4.7 SmGPR-2 is expressed in the subtegumental nerve plexus of schistosomula and adult schistosomes To investigate the role of histamine, the tissue localization of SmGPR-2 was examined.76 For this, a polyclonal anti-SmGPR-2 antibody was used. 7-day-old schistosomula and adult S. mansoni were probed with the antibody, after which a FITC-labelled secondary antibody was added. Furthermore, muscles and cytoskeletal elements were labeled by treating some of the animals with rhodamine- conjugated phalloidin.143,144 SmGPR-2 immunoreactivity was found in the subtegumental region of the schistosomula (Figure 27A, B).76 No co-localization of the receptor and muscle was observed (Figure 27C).76 This indicates that SmGPR-2 is not associated with the musculature, but most likely with the nervous system. The distribution pattern of SmGPR-2 in adults is similar to that of schistosomula. However, The level of SmGPR-2 expression was lower and less widespread in adults compared to schistosomula. These results are in agreement with those of the qPCR studies (section 4.2). In adult worms, SmGPR-2 expression was detected in the peripheral neuronal plexus of the subtegumental layer (Figure 27D, E).76 An important observation is that the localization of SmGPR-2 in this area resembles that of histamine (section 5.4.1). Adult female schistosomes that

65 were probed with an anti-histamine and anti-SmGPR-2 antibody showed juxtaposed signals in the nerve plexus (Figure 27F).76 It can thus be suggested that endogenously released histamine could activate SmGPR-2. However, no co-localization was observed, which indicates that SmGPR-2 and histamine associate with different neurons. This observation may support the hypothesis that histamine acts through SmGPR-2 to regulate the activity of different neurotransmitters in the subtegumental region. For example cholinergic, peptidergic and serotonergic neurons that are present in this area could be modulated in this way.

Figure 27: Localization of SmGPR-2. Seven-day old schistosomula (A–C) and adult S. mansoni (D–F) were treated with anti- SmGPR-2 antibodies, after which FITC labeled secondary antibodies were added. SmGPR-2 immunoreactivity (green) was detected in the subtegumental layer of schistosomula (A–B). Muscle and cytoskeletal elements were labeled with (TRITC)- labeled phalloidin (red). SmGPR-2 is enriched in neurons with no apparent co-localization in the muscles (C). In adult female schistosomes, SmGPR-2 immunoreactivity was detected in the neuroplexus of the subtegumental layer (D–E). Adult female schistosomes that were probed with an anti-histamine (red, open arrows) and anti-SmGPR-2 (green, solid arrows) antibody showed juxtaposed signals in the nerve plexus (F). No co-localization of these two antibodies was observed. Asterisk mark the non-specific fluorescence.76

In summary, SmGPR-1 and SmGPR-2 were demonstrated to be selectively activated by histamine. SmGPR-1 was shown to be expressed in the musculature and tegument of schistosomula and adult worms. SmGPR-2 is expressed near histamine-containing neurons and is enriched in the subtegumental nerve plexus in both adult worms and schistosomula. Previous studies showed that histamine modulates schistosome motility.123 The detection of multiple histaminergic neurons in the peripheral plexus that support the body wall musculature corroborates these earlier findings.76 The location of SmGPR-1 and SmGPR-2 suggest that they could contribute to schistosome motility. No colocalization experiments have been performed which could prove the colocalization of SmGPR-1 and SmGPR-2. However, it is possible to hypothesize that the observed effects of motility could be a result of SmGPR-1 activation, which is expressed on the body wall muscles.77 Alternatively, they could be achieved by regulation of neuromuscular circuits through SmGPR-2. Since histamine-containing fibers were detected in the suckers of S. mansoni, an additional role for histamine could be in the musculature of these structures. The presence of SmGPR-1 in both suckers suggest that these effects are regulated (partially) by this receptor.

These findings begin to unravel the mode of action of histamine in S. mansoni. Further research should be undertaken to investigate whether histamine is able to activate other member of the SmGPR clade. Further work is also required to establish whether S. mansoni synthesizes histamine endogenously or if it is obtained from the host. Section 6.4 analyzes the pharmacology profile of SmGPR-2. The next section discusses the functional analysis and characterization of SmGPR-3.

5.4.8 SmGPR-3 is activated by dopamine and epinine SmGPR-3 (Smp_043290) belongs to the same clade as SmGPR-1 and SmGPR-2. As discussed in section 5.4.2, this SmGPR clade is characterized by the substitution of the highly conserved aspartate D3.32 of TM3 to an asparagine. However, the only exception to this is SmGPR-3, since the aspartate D3.32 is conserved in this receptor. This section analyzes functional expression studies of SmGPR-3. Then, the structural analysis of SmGPR-3 is discussed. To gain insight into the potential functions of SmGPR-3, the tissue localization of SmGPR-3 is analyzed. Lastly, in vitro motility assays are described to determine whether various SmGPR-3 antagonists and agonists influence worm motility.

SmGPR-3 cDNA was cloned from adult S. mansoni by RT-PCR.80 Functional expression studies were conducted in S. cerevisiae strain YEX108.79,97,110 This is a histidine auxotrophic strain that expresses a HIS3 reporter gene under the control of a FUS1 promoter.109 It also contains an integrated copy of a chimeric Gα gene in which the first 31 and last five codons were of human Gαq instead of those of native yeast.110 Strains carrying Gαi2, Gα12, Gαo, Gαs yielded lower or no receptor activity. The activation of the recombinant GPCR was quantified by measuring yeast growth in a selective medium, using a fluorometric Alamar Blue assay. Cells that were transformed with either SmGPR-3 or empty vector (mock) were treated with biogenic amines (2x10-4 M) (adrenaline, dopamine, epinine, histamine, noradrenaline, octopamine, serotonin, tyramine) (Figure 28A).80 The results revealed that SmGPR-3 was selectively activated by dopamine as well as by its metabolite epinine. Adrenaline, noradrenaline and metanephrine were also recognized by the receptor, but resulted in a lower response. Partial constitutive activity of SmGPR-3 was observed without the presence of an agonist. The receptor could be further activated by catecholamines, but not significantly by other biogenic amines. The response to epinine and -5 -5 dopamine were shown to be dose-dependent with an EC50 of 2.85x10 M and 3.1x10 M, respectively (Figure 28B).80 The potential binding site(s) of dopamine and epinine can be analyzed by generating a homology model of SmGPR-3. The next section analyzes the structural organization of SmGPR-3.

Figure 28: Functional expression studies of SmGPR-3 in yeast. A) SmGPR-3 cDNA or empty plasmid (mock) was expressed in S. cerevisiae strain YEX108 and grown in a histidine-deficient medium. The cells were treated with one of the biogenic amines (2x 10-4 M) or vehicle (ND). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth (RFU, relative fluorescence units). SmGPR-3 exhibited constitutive activity, but was further activated by catecholamines (dopamine, DA; epinine, EPN; adrenaline, A; noradrenaline, NA; and metanephrine (not shown). No activity was measured in the cells that were treated with other biogenic amines (octopamine, OA; serotonin, 5-HT; tyramine, TA; histamine, HA). B) Experiments were repeated using various concentrations of epinine and dopamine.80

5.4.9 The binding pocket of SmGPR-3 does not resemble that of mammalian dopamine receptors To gain more insight into the structural organization of SmGPR-3, a homology model of the receptor was generated (Figure 29A).80 The human b2-adrenergic receptor was chosen as a template. This hypothetical structure was used for docking-simulations (Figure 29B).80 Previous studies have shown that D3.32 in TM3 is important for anchoring the protonated amino group of the ligand in catecholamine receptors.126,128,153–157 Furthermore, three serines in TM5 (S5.42, S5.43 and S5.46) undergo interactions with the hydroxyl groups on the catechol ring. From these residues, S5.42, S5.43 and D3.32 are conserved in SmGPR-3. The results showed that D3.32 is involved in binding of dopamine, while the serines of TM5 most likely are not. Instead, potential binding sites of

69 dopamine (Figure 29C) are located in TM7 and TM2. Surprising differences in the potential binding site compared to that of mammalian dopamine receptors were discovered. For example, the conserved aromatic residues of TM6 (F6.51, F6.52) appeared not to be involved in any interactions with the ligand. Furthermore, the analysis revealed that R2.64 of TM2 directly interacts with dopamine, while in mammalian receptors this residue is not conserved. This residue locates near the extracellular junction of TM2, which is an important component of the binding site of antagonists in the human D3 receptor.154 For this reason, this region may also be of importance for the pharmacology and function of SmGPR-3.80 The fact that R2.64 is conserved in all members of the SmGPR clade but not in receptors of the mammalian host further highlights that this region could potentially be a target for selective antischistosomal drugs. When the experiments were repeated using epinine (Figure 29D), the results revealed that this ligand binds to the same binding pocket as dopamine. Moreover, the residues of SmGPR-3 that are involved in interactions with dopamine are also involved with the binding of epinine. These residues are R2.64 of TM2, D3.32 of TM3 and T7.39 and Y7.43 of TM7. These data must be interpreted with caution due to the low sequence homology of the receptor in comparison to the b2-adrenergic template. These differences between the schistosome and host receptors have not yet been tested by mutagenesis and functional analyses.

These results revealed important differences between the binding pocket of mammalian host receptors and that of SmGPR-3. These differences may be exploited to identify selective antischistosomal drugs.158,159 For example, target-based in silico screening could be applied. Furthermore, future investigations could test the identified residues by mutagenesis and functional studies. The next section analyzes the tissue localization of SmGPR-3.

Figure 29: Homology model of SmGPR-3 and ligand docking simulations A) Homology model of SmGPR-3 with the human b2-adrenergic receptor as a template. Dopamine (DA) is the ligand that was used. The 7 transmembrane (TM) positions that are predicted are shown, even as the extracellular loops (ECL1, 2, 3). The intracellular helix is marked (helix 8). B) Predicted binding site of dopamine C) Structure of dopamine D) Structure of epinine.80

5.4.10 SmGPR-3 is located in the nervous system of S. mansoni To study the tissue localization of SmGPR-3 and to gain insight into the role of the receptor, a specific antibody was produced.78 Subsequently, in situ immunolocalization studies were performed on adult and larval S. mansoni. The various life stages were probes with anti-SmGPR3, after which a FITC-labeled secondary antibody was introduced. In addition, muscles and cytoskeletal elements were labeled by treating the various S. mansoni life-stages with TRITC- conjugated phalloidin.144 The results revealed that SmGPR-3 is mainly present in the nervous system of S. mansoni adults and larvae. Remarkably, the tissue distribution of the receptor in cercariae resembles that of dopamine itself, which was described in a previous study on S.

71 japonicum and S. mansoni.160 These catecholamine-containing neurons were found to be localized in the posterior transverse commissure and the major longitudinal nerve cords. SmGPR-3 was expressed in these region as well. It can thus be suggested that dopamine that is released from these neurons activates this receptor. Consequently, SmGPR-3 may regulate inter-neuronal dopamine signaling within the central nervous system of cercariae. In adult worms, this receptor was found to be more widely expressed. Immunoreactivity was detected in parts of the peripheral nervous system and in the central nervous system. This suggest that SmGPR-3 plays a more diverse role in adult schistosomes. The receptor was found to be expressed in the cerebral ganglia and the main nerve cords. Additionally, immunoreactivity was detected in the peripheral innervation of the body wall muscles and suckers. These results indicate that SmGPR-3 plays important roles in and outside the central nervous system of adult S. mansoni. Since this receptor is present in the fibers that innervate the parasite musculature, it is suggested that it controls muscle function. Hence, it could conceivably be hypothesized that this is one of the routes of dopaminergic motor control in S. mansoni.

Another receptor that was previously identified as a dopamine GPCR is SmD2 (section 5.3).66 This receptor is not part of the SmGPR-like clade, but it shares sequence homology with D2-like receptors. This receptor is not present in the central nervous system, but is present in the body wall muscles. SmGPR-3 is also expressed in the body wall, but there was no apparent co-localization of SmGPR-3 and muscle, which was labelled by the use of phalloidin. This indicates that SmGPR- 3 is associated with neuronal elements instead of the muscles. These findings suggest that there multiple dopaminergic mechanisms that control worm motility. SmGPR-3 could mediate an indirect neuronal pathway, while SmD2 could act directly on the body wall muscle. The former could control neuronal input to the musculature, most probably by regulating the signaling activity or release of another (neuro)transmitter. To test whether SmGPR-3 is involved in dopaminergic motor control, in vitro motility assays were performed. The next section analyzes these assays.

5.4.11 Dopamine, epinine and dopaminergic substances induce strong effects on the motor activity of larvae Previous studies have shown that when S. mansoni is treated with dopamine, relaxation of the body wall muscles occurs.106 This effects seems to be receptor-mediated, but little information is known about the mechanism. SmD2 is likely one of the receptors that contribute to the described effects of dopamine on the musculature of S. mansoni (section 5.3).66 The tissue localization of SmGPR- 3 suggest that this receptor may also contribute to the observed motor effects of dopamine. To determine whether various SmGPR-3 antagonists and agonists influence worm motility in vitro motility assays were performed.80

In order to quantitate motility, individual schistosomula were treated with various drugs and monitored using a microscope, a video camera and imaging software. Untreated animals functioned as controls (Figure 30A).80 Schistosomula undergo contraction and elongation, which causes a decrease or increase in body length up to 20%. When schistosomula were treated with epinine or dopamine (100 µM), this behavior changed. Treatment with epinine resulted in virtual paralysis, whereas dopamine decreased the frequency and amplitude of the contractions. The assay was repeated with various concentrations of epinine and dopamine and the frequency of length changes was reported (Figure 30B).80 The results revealed that treatment with each amine (≥ 10 µM) resulted in a dose-dependent inhibition of schistosomula motility. Therefore, these findings further support the idea of dopamine having inhibitory effects on schistosome motility. In previous studies, an overall increase in body length of adult worms was reported.161,162 However, this was not observed in this study on larvae. This could be because SmGPR-3 is more widely distributed in adults (section 5.4.10). In comparison to adults, the expression distribution of the receptor is more restricted to the central nervous system in larvae. It can be hypothesized that the differences in expression patterns in the two life stages cause different responses to dopamine. Alternatively, the observed differences could be a result of differences in experimental protocols.

Figure 30: Effects of dopamine, epinine and other drugs on schistosomula motor activity. A) Schistosomula were treated with epinine (EPN) or dopamine (DA) (100 µM) or vehicle (CT). After 5 minutes (RT) the schistosomula were monitored using a microscope, a video camera and imaging software. B) The assay was repeated with different concentrations of dopamine and epinine (10-7 M – 10-4 M) or vehicle (CT). Motility is the frequency of length changes/minute. C) Schistosomula were incubated with various drugs or without any test substances (CT). Subsequently, motility of the animals was measured. Promethazine, PRMZ; epinine, EPN; flupenthixol, FLPX; dopamine, DA (50 µM). Haloperidol, HLRD; adrenaline, A; metanephrine; MTN (500 µM).80

In addition to epinine and dopamine, drugs that were demonstrated to have activity towards SmGPR-3 (section 6.4.2) were tested in the same motility assay. Among these were haloperidol, adrenaline, flupenthixol, metanephrine and promethazine. All drugs, except haloperidol, induced significant changes in motility (Figure 30C) compared to the controls.80 Since exogenous applied dopamine results in paralysis of larvae, it was predicted that antagonists of SmGPR-3 results in hyperactivity. However, promethazine and flupenthixol induced inhibition of motor activity. It may be the case that these drugs interact with multiple receptors in S. mansoni. For example, they could interact with SmD2, which is another dopamine receptor that was identified in S. mansoni (section 5.3).66 Moreover, flupenthixol and promethazine were shown to inhibit SmGPR-2 (section 6.4.1).76 When the larvae were treated with adrenaline, paralysis was observed. Remarkably, metanephrine, which is structurally similar to adrenaline, caused hyperactivity. It may be the case that

74 metanephrine competes with dopamine, since weak agonistic effects were observed when it interacted with recombinant SmGPR-3. This, in turn, may result in a decrease in endogenous signaling. Overall, these results indicate that catecholamine metabolites and other dopaminergic substances induce strong effects on the motor activity of larvae. Further work is required to establish the mode of action and to identify additional selective inhibitors that could disrupt worm motility.

To conclude, SmGPR-3 is activated by dopamine as well as other catecholamines. However, this receptor is not similar to any mammalian dopamine receptor. The sequence homology as well as the predicted binding site differ from those of mammalian receptors. Immunolocalization studies revealed that SmGPR-3 is predominantly expressed in the nervous system of S. mansoni. Therefore, it is hypothesized that this receptor plays an important role in neuromuscular and neuronal signaling. Section 6.4.2 analyzes the drug profile of SmGPR-3. The next section discusses the functional analysis and characterization of serotonin receptor Sm5HTR.

5.5 Serotonin receptor Sm5HTR As described in section 1.2, the central nervous system is involved in the control of muscle function and movement and thus is crucial for the survival of the parasite. Therefore, GPCRs that are expressed in the central nervous system of S. mansoni have strong potential for anti-schistosomal drug targeting. As discussed in section 3.2, serotonin receptor Sm5HTR (Smp_126730) is widely distributed in the central nervous system of S. mansoni. Serotonin is myoexcitatory in S. mansoni, since it stimulates motor activity and increases muscle contraction.31 There is, however, little understanding of the mechanism of serotonin. This section discusses the sequence analysis, functional expression studies and immunolocalization of Sm5HTR. Furthermore, the effects of various serotonin antagonists and agonists on larval body movement are analyzed.

5.5.1 Sm5HTR contains residues that are directly involved in the binding of serotonin Sm5HTR has all hallmark features of class A GPCRs, including the 7 TM topology, the NPxxY motif of TM7 and the DRY-motif at the end of TM3. Sequence analysis showed that Sm5HTR is distantly related to 5HT7 from other species. Furthermore, residues that are known to be involved in serotonin binding and activity are present. Compared to two human serotonin receptors,163,164 all

75 residues of the serotonin binding pocket were identified in Sm5HTR except one. These include D1153.32, T1203.37 and A1995.46, which directly interact with serotonin in the human receptors. However, not all residues in the core binding site are conserved. Sm5HTR has an alanine (A1955.42) in the place of a conserved serine, which is involved in the interaction with the indole ring of serotonin. Even though the binding pocket is mostly conserved, the overall level of homology is less than 40%. Moreover, a lot of residues that surround the core binding pocket are not conserved in the schistosome receptor. These include residues that have been shown to be involved in drug binding to human receptors.163,164 An important site for conformational activation, the canonical glutamate of TM6 that is part of the ionic-lock motif, is substituted with an aspartate in the schistosome receptor (D3006.30). Another receptor that does not contains this conserved residue is SmGAR (section 5.2), which has an alanine (Ala8486.30) instead of the critical glutamate of TM6.94

5.5.2 Sm5HTR is activated by serotonin and signals through an increase in cytosolic cAMP Sm5HTR was heterologously expressed in HEK293 cells to test for receptor activity.31 A fluorescence-based assay was used to measure changes in cytosolic Ca2+.114 The results showed there was no effect of the schistosome receptor on cytosolic calcium levels. In contrast, the results of a cAMP signaling assay showed that serotonin significantly increased the cAMP level in cells that expressed Sm5HTR (Figure 31A).31 Furthermore, the receptor was activated in cells that were treated with o-methyl-serotonin and mammalian serotonergic agonists. Cells that were treated with other biogenic amines did not result in significant receptor activation, suggesting that Sm5HTR is selectively activated by serotonin and related compounds. The effect of serotonin as well as o- 31 31 methyl-serotonin are dose-dependent (Figure 31B–C). . The observed EC50 value of serotonin is 61.7 nM and of o-methyl-serotonin 72.2 nM. The mechanism of the receptor is in accordance with previous studies of serotonin in schistosomes. Research has shown that when crude worm extracts are treated with serotonin, it results in the stimulation of adenylate cyclase.165,166 Moreover, cAMP is thought to be the predominant mediator of serotonin signaling in all flatworms. Overall, these results showed that Sm5HTR is activated by serotonin and a related compound, and signals through an increase in cAMP when expressed in HEK293 cells. The next section analyzes the effect of serotonin antagonists and agonist on larval motility.

A B

Figure 31: Functional analysis of Sm5HTR. A) Sm5HTR was expressed in HEK293 cells. The cells were treated with common serotonergic antagonists and agonists as well as biogenic amines (10-4 M). Following incubation, cells were lysed. These cell lysates were assayed for cAMP by using a competitive immunoassay. The control cells (mock) were transfected with empty plasmid and were used to normalize the data. B) Experiments were repeated with various concentrations of serotonin and C) with o-methyl-serotonin. Acetylcholine, Ach; adrenaline, A; buspirone, BUS; chlorpromazine, CHP; cyproheptadine, CPH; dopamine, DA; 8-Hydroxy-DPAT, DPAT; histamine, HA; metanephrine, MTN; o-methyl-serotonin, methyl-5HT; mianserin, MIAN; octopamine, OA; serotonin, 5HT; tryptamine, Trp; tyramine, TA.31

5.5.3 Sm5HTR plays a crucial role in endogenous motor control in schistosomes Since serotonin is known to influence schistosome movement, it was tested whether Sm5HTR plays a role in this activity. Young schistosomula (four to six days) were tested for motor activity after treatment with various concentrations of o-methyl-serotonin or serotonin in the absence or presence of antagonists. The results showed that o-methyl-serotonin as well as serotonin dose- dependently stimulated larval motility. The effect of serotonin on larval motility was almost completely inhibited when the cells were treated with serotonin and either mianserin or cyproheptadine (100 µM), which are serotonergic antagonists. This result is in agreement with a receptor-mediated mechanism. To test whether Sm5HTR plays a role in motor control, RNA

77 interference (RNAi) studies were performed. The results revealed that the frequency of body movement of the schistosomula that were treated with Sm5HTR siRNA was on average 80% less compared to the controls (Figure 33A).31 Nearly complete knockdown of the receptor was verified by reverse transcription (RT)-qPCR analysis (Figure 33B).31 The siRNA treated schistosomula did not show elongation and shortening of their body, and appeared to be more round in shape compared to the controls (Figure 33C).31 The experiments were repeated in the presence of serotonin (100 µM). The results showed that whereas the controls were still responsive to serotonin, the Sm5HTR siRNA-transfected schistosomula were not. This indicates that Sm5HTR is a required for normal larval motility. Motility assays were also performed with adult schistosomes. The results revealed that schistosomes treated with Sm5HTR siRNA were significantly less motile compared to the controls. Knockdown of Sm5HTR in adults schistosomes was confirmed by reverse transcription (RT)-qPCR analysis. Taken together, these results show that Sm5HTR is equally important for motor activities in adult schistosomes and in schistosomula.

Figure 32: The effect of serotonin on larval motility. Schistosomula were treated with different concentrations of A) serotonin (5HT) or B) o-methyl-serotonin. An imaging assay was used to quantify the frequency of body movements/min.167 C) The experiments were repeated with co-treatment of serotonin and a serotonergic antagonist (each 10-4 M). Cyproheptadine, CPH; mianserin, MIAN.31

Figure 33: RNAi studies in schistosomula. Schistosomula were treated with either Sm5HTR siRNA (50 nM) or scrambled (irrelevant) siRNA (50 nM). A mock-transfected control was also prepared. A) After 8 days the motility assays were performed. An imaging assay was used to quantify the frequency of body movements/min.167 B) Knockdown of Sm5HTR was confirmed by reverse transcription (RT) -qPCR analysis. Primers that targeted Sm5HTR and a-tubulin (housekeeping gene) were used. C) Images of the animals that were transfected or transfected with either scrambled siRNA or Sm5HTR siRNA. Elongation and shortening is visible in the controls, whereas this is not visible in the more round (in shape) schistosomula that were treated with Sm5HTR siRNA.31

Since Sm5HTR is widely expressed in the nervous system of S. mansoni, it is likely that serotonin acts through this receptor to stimulate neuromuscular and/or interneuronal signaling and by doing so stimulates movement indirectly. The hypoactive phenotypes that were observed when the animals were treated with either a serotonin antagonist or Sm5HTR siRNA could then be explained by loss of signaling.

In summary, Sm5HTR is a specific serotonin receptor and its activation results in an increase in cAMP. Treatment with exogenous serotonin causes dose-dependent hyperactivity of schistosomula and adults worms. This effect is mediated by at least Sm5HTR. The results showed that Sm5HTR is required for endogenous motor control in S. mansoni. Therefore, Sm5HTR is an interesting potential drug target. The pharmacological profile of Sm5HTR is analyzed in section 6.5.

5.6 Summary This chapter analyzed all the S. mansoni receptors that are deorphanized so far. These included the glutamate receptor SmGluR, which signals through a different pathway than mammalian mGLuRs.67 This receptor potentially plays a role in muscle function as well as egg production and control. SmGAR is a cholinergic receptor that stimulates motility in schistosome larvae and exhibits constitutive activity.94 SmD2 is S. mansoni receptor that is selectively activated by dopamine.66 The receptor signals through stimulation of cAMP, which is atypical for a D2-like receptor. SmD2 is expressed in the somatic muscles and therefore suggested to play a role in parasite motility. SmGPR-3 is a dopamine receptor as well and is associated with neuronal elements instead of the muscles.80 It is suggested that SmGPR-3 controls neuronal input to the musculature, while SmD2 may act directly on the body wall muscle. SmGPR-3 belongs to the same schistosome-specific clade as SmGPR-1 and SmGPR-2.76 These two receptors are both selectively activated by histamine.76,79 Histamine may play an important role in host-parasite interactions. Both histamine receptors may be involved in the control of the musculature. This could be achieved by direct activation of SmGPR-1, which is expressed on the body wall muscles, or indirectly by regulation of neuromuscular circuits through SmGPR-2. Lastly, Sm5HTR is a specific serotonin receptor and its activation results in an increase in cAMP.31 This receptor is required for endogenous motor control in S. mansoni. It must be considered that these receptors may be activated by schistosome-specific ligands that are structurally similar to the assigned ligands.

The deorphanization of these GPCRs revealed potential antischistosomal drug targets. This is supported by their potential roles in the control of muscle function and movement, which are processes that are crucial for the survival of the parasite. Furthermore, the identification of endogenous ligands for schistosome GPCRs helps unravelling the complex biology of S. mansoni. The next chapter analyzes the pharmacological profiles of the deorphanization S. mansoni GPCRs and discusses the possibility of repositioning promethazine as an antischistosomal drug.

6. Pharmacology of GPCRs in S. mansoni GPCRs comprise the largest superfamily of transmembrane receptors in the genome of S. mansoni.15,75 So far, this paper has shown that all major GPCR subfamilies, and a Platyhelminth- Specific Family, are present in S. mansoni (Chapter 2). Furthermore, GPCRs play a role in the complex sex interplay of schistosomes (Chapter 3).17,22 These receptors contribute to non-gonad, pairing-dependent processes as well as to gonad-specific functions. Targeting these receptors may result in disruption of crucial activities and interference of the normal life cycle of S. mansoni. Chapter 4 showed that various GPCRs are upregulated in parasitic life-stages. Various GPCRs are upregulated during the development of early schistosomula. These GPCRs are potential candidates for drug targeting since drugs that target these receptor have potential activity against various schistosome life stages. This is especially important since juvenile schistosomes are refractory to the currently available drug to treat Schistosomiasis ((PZQ).8 Even though these findings highlight the importance of GPCR signaling in S. mansoni, only a small part of these receptors have been functionally characterized. The S. mansoni receptors that are deorphanized were discussed in Chapter 5. The results revealed receptors with potential functions in the muscle and tegument of S. mansoni and neuromuscular and neuronal signaling. These results contribute to the identification of molecules and key pathways that are crucial to parasite biology and reveal promising candidate targets for antischistosomal drugs. In addition, the diversity of GPCRs is large among species and they are able to bind distinct ligands.17 For this reason, tailor-made drugs can be developed that reduce the possibility of host toxicity. However, knowledge of drug profiles of schistosome GPCRs is limited.168 This chapter analyzes the pharmacological profiles of deorphanized schistosome GPCRs. Section 6.5 analyzes the pharmacological divergence of a Sm5HTR from the closest human homolog. The last section discusses the efficacy of promethazine against S. mansoni ex vivo and in a mouse model of schistosomiasis.

6.1 Pharmacological profile of glutamate receptor SmGluR

SmGluR (Smp_128940) contains non-canonical LBDs for a glutamate-like receptor (Figure 6).15 This receptor was shown to selectively respond to glutamate in ligand-binding assays (section 5.1).67 Since most mGluRs inhibit adenylyl cyclase and decrease cAMP levels,89 receptor activity was tested by measuring changes in the level of intracellular cAMP. HEK-293 cells expressing SmGluR were treated with various classical mammalian glutamate receptor agonists and

81 antagonists (Figure 34A).67 Only LY341495, which is a selective antagonist of the group II metabotropic glutamate receptors, could interact with the receptor and produced a reasonable dose- dependent inhibition of receptor activity (Figure 34B-C). Since SmGluR is responsive to glutamate, but its activity cannot be modulated by the majority of the classical (mammalian) agonists and antagonist tested, it is suggested that SmGluR has a different pharmacological profile than mammalian (host) glutamate receptors.

Figure 34: Drug profile of glutamate receptor SmGluR. A) SmGluR was expressed in HEK-293 cells and treated with various classical mammalian glutamate receptor agonists (10-4 M). A competitive immunoassay was performed to measure cAMP levels. A mock-transfected control was also prepared to use for the normalization of the data. B) The same cells were treated with different concentrations of LY341495 in the presence of glutamate (100 µM). LY341495 was able to inhibit the receptor in a dose-dependent manner. C) Structure of LY341495.67

In summary, SmGluR is a metabotropic glutamate receptor in S. mansoni. The receptor is selectively activated by glutamate when expressed in a heterologous expression system. The mechanism of signaling (section 5.1) as well as the pharmacological profile is different compared to mammalian mGluRs, suggesting that the receptor is diverged early in evolution. Since the receptor potentially influences muscle function indirectly and may be involved in the regulation of egg production and control (section 5.1), targeting this receptor could result in the elimination of the parasite from the host. This further highlights the potential of SmGluR as an antischistosomal

82 drug target. Future studies could investigate whether compounds could be developed that selectively target schistosome glutamate receptors, which would reduce the reduce the possibility of host toxicity. The next section analyzes the pharmacological profile of SmGAR.

6.2 Pharmacological profile of acetylcholine receptor SmGAR 6.2.1 Promethazine and atropine act as inverse agonists on SmGAR SmGAR is constitutively active GPCR that responds to acetylcholine and likely plays an important role in early parasite migration (section 5.2).94 Since it is constitutively active, inverse agonists can be utilized to characterize its pharmacology.169 Inverse agonist can reduce the basal activity of the receptor because they preferentially bind to and stabilize the inactive receptor conformation, thus displaying a negative intrinsic activity. Research has shown that compounds that are known as competitive antagonists, can act as inverse agonists when they are tested on receptors that are constitutively active.

To assess the pharmacology of SmGAR, yeast expressing SmGAR were treated with various compounds with known anti-cholinergic activity.94,170 For a description of the yeast functional assay that was performed, see section 5.2.97,110 Various concentrations of pirenzepine, atropine or promethazine were added to the cells without acetylcholine. The results showed that promethazine as well as atropine dose-dependently decreased the elevated basal activity of SmGAR, whereas pirenzepine did not have a significant effect up to a concentration of 100 µM (Figure 35).94 Pirenzepine and atropine are both classical muscarinic receptor antagonists, while promethazine is an antagonist at muscarinic cholinergic and histamine H1 receptors.170 Cells were plated in non- selective media with antagonist (1 mM) to ensure that the effects were not caused by cytotoxicity. The results showed that there was no growth inhibition.

Figure 35: Promethazine and atropine have inverse agonist activity towards S. mansoni receptor SmGAR. SmGAR was expressed in yeast. The cells were treated with different concentrations of either pirenzepine, promethazine or atropine in the absence of acetylcholine to test for inverse agonism. Promethazine and atropine significantly decreased the basal activity of SmGAR in a dose-dependent manner (A-B). In contrast, pirenzepine did not have any effect on the basal activity of SmGAR.94

These results showed that promethazine as well as atropine dose-dependently abrogated the elevated base activity of SmGAR. Atropine was shown to be the most potent compound. Since the inhibition occurred without the presence of acetylcholine, the compounds acted as inverse agonists. It is possible that pirenzepine was not able to recognize the inactive receptor, since it did not have a significant effect. These findings further support the idea that SmGAR has intrinsic constitutive activity. In recent years, there has been an increasing interest in the therapeutic benefits of inverse agonists.149,169 The results of the described experiments also show that they have potential for GPCR and anthelmintic drug discovery. In future investigations, inverse agonist screens as well as antagonists assays can be performed in order to identify selective SmGAR inhibitors. Since motor function is crucial to the survival of S. mansoni, SmGAR is an interesting potential drug target. Section 6.6 describes the efficacy of promethazine against S. mansoni ex vivo and in a murine model of schistosomiasis.

6.3 Pharmacological profile of dopamine receptor SmD2 6.3.1 A mammalian dopamine agonist acts as an antagonist on SmD2 SmD2 is a GPCR that is selectively activated by dopamine (section 5.3).66 It is suggested that dopamine plays an important role in neuromuscular control,106 which is why SmD2 is an interesting potential drug target.66 To assess the effects of various classical ligands that target biogenic amine receptors, S. cerevesiae strain CY13399 was transfected with SmD2 and grown in histidine- deficient media. These cells were treated with 100 µM dopamine and 100 µM of test agent or vehicle. An Alamar blue assay was used to quantify receptor activity based on cell growth. Remarkably, only chlorpromazine (cholinergic/dopaminergic antagonist) and apomorphine (classical D1/D2 dopaminergic agonist) inhibited the activity (>50%) of SmD2 that was induced by dopamine (Figure 36A).66 The experiments were repeated with various concentrations of the drugs (Figure 36B).66 The results revealed that responses caused by apomorphine and chlorpromazine were both dose-dependent. The observed effects were shown not to be caused by drug-induced toxicity. It is striking that a classical mammalian dopamine agonist (apomorphine) acted as a high potency SmD2 antagonist, while classical antagonists sulpiride and SCH23390 had little to no effect. Spiperone and haloperidol, which are both classical antagonists of dopamine receptors, were able to inhibit the response to 50%. A previous study showed that spiperone blocks the effect of dopamine on the musculature of S. mansoni.106

Figure 36: Drug profile of S. mansoni receptor SmD2. A) SmD2 cDNA was expressed in S. cerevisiae strain CY13399 and grown in a histidine-deficient mediumThe cells were treated with 100 µM dopamine and 100 µM of test agent or vehicle (control). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth. Controls were used that were treated with dopamine but without antagonist to normalize the data. Apomorphine, APOM; chlorpromazine, CPZ; haloperidol, HAL; propranolol, PROP; SCH23390; spiperone, SPIP; sulpiride, SULP. B) Experiments were repeated with various concentrations of test compound and a constant amount of dopamine (100 µM ).66

These results showed that SmD2 has an unusual pharmacological profile. Even though the receptor responded to some classical dopamine agonists, their relative affinities differed from mammalian D1 or D2 profiles. Most notable, a classical mammalian dopamine agonist acted as an antagonist with high potency on SmD2. Since SmD2 is likely to play a role in controlling worm motility, it could potentially be an important antischistosomal drug target. The uniqueness of the drug profile further highlights the potential of SmD2 as a drug target, since schistosome specific drugs could potentially be developed. The next section analyzes the drug profiles of members of the SmGPR clade.

6.4 Drug profiles of SmGPR-like receptors 6.4.1 SmGPR-2 has an unusual drug profile SmGPR-2 is member of the SmGPR clade, which exist in schistosomes but not in the mammalian host. This receptor was shown to be selectively activated by histamine (section 5.4.6).76 Histamine was shown to stimulate motility in S. mansoni.123 Furthermore, administration of promethazine to schistosomes resulted in paralysis, which could be reversed by histamine. This demonstrates that importance of histamine receptors in S. mansoni as well as the potential of these receptors for drug targeting. SmGPR-2 is likely to modulate neuromuscular circuits, potentially resulting in the aforementioned effect of histamine on schistosome motility.76 Yeast functional assays (section 5.4.6) were performed to gain insight into the pharmacological properties of SmGPR-2.

The results revealed that SmGPR-2 was activated by a histamine metabolite named 1-methylhistamine (1-methylHA) and strongly inhibited by promethazine (Figure 37C), a common histaminergic antagonist (Figure 37A).76 The inhibition by promethazine was dose- dependent and a concentration of 10-4 M promethazine resulted in loss of all receptor activity (Figure 37B).76 To test for drug-induced toxicity, the experiments were repeated in medium supplemented with histidine, which results in cell growth irrespective of activation of SmGPR-2. The results showed that there was no drug-induced toxicity, since normal cell growth was detected when the cells were in the presence of promethazine and histidine (Figure 37B).76 Other mammalian histamine antagonists (ranitidine, cimetidine and diphenhydramine) were tested as well, but these were not able to inhibit the receptor (Figure 38).76 However, addition of flupenthixol (P < 0.001), buspirone (P < 0.01) or cyproheptadine (P < 0.001) did result in receptor activity inhibition (Figure 38).76 Buspirone and flupenthixol are classical antagonists of serotonin and dopamine receptors, respectively. Remarkably, these are not known to have antihistamine properties. In contrast, cyproheptadine has been demonstrated to have antiserotonin- antihistaminic properties in vertebrates. Treatment of sulpiride (dopamine antagonist) as well as mianserin (serotonergic/adrenergic/histaminergic antagonist) did not result in inhibition of SmGPR-2 activity (Figure 38).76

Figure 37: Pharmacological studies of SmGPR-2. A) SmGPR-2 was expressed in yeast strain YEX108 and is activated by histamine (HA) or 1-methylhistamine (1-metHA). SmGPR-2 cDNA or empty plasmid (mock) was expressed in S. cerevisiae strain YEX108 and grown in a histidine-deficient medium. The cells were treated with HA or 1-metHA (100 µM) or vehicle (ND). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth (RFU, relative fluorescence units). B) These experiments were repeated but then in the presence of different concentrations of promethazine or vehicle (–). The control lacked promethazine as well as agonist (ND). Inhibition of SmGPR-2 activity by promethazine is dose-dependent. Experiments were repeated in the presence of promethazine and histamine to test for drug-induced toxicity (+ve). C) Structure of promethazine76

Figure 38: SmGPR-2 has an unusual drug profile. Histamine (HA) and a test antagonist (100 µM, except FLP: 10 µM) were added to clones of SmGPR-2. The control was treated with histamine but no antagonist (dotted line). Tested compounds were buspirone, BUS; cimetidine, CMT; cyproheptadine, CPH; diphenhydramine, DPH; flupenthixol, FLP; mianserin, MNS; promethazine, PMZ; ranitidine, RNT; sulpiride, SLP.76

In summary, promethazine was the only histaminergic that produced a significant inhibition of SmGPR-2 activity. To a lesser extent, cyproheptadine inhibited histamine induced SmGPR-2 activity, which is a mixed antagonist that has both histaminergic and serotonergic activity. Interestingly, promethazine has been shown to induce spasmodic contractions in adult schistosomes followed by a reduction in motor activity.171 The pharmacological profile of SmGPR- 2 is unusual because histamine activates the receptor but various classical anti-histamines have no effect. Interestingly, the receptor also does not conform to other known histaminergic receptors

89 structurally (section 5.4). Therefore, it is plausible that SmGPR-2 belongs to a novel type BA receptor. This combination of findings highlights the potential of SmGPR-2 as a schistosomiasis drug target. Future studies will need to be undertaken to confirm if their drug profiles are parasite- specific. In addition, further work is required to investigate the pharmacological profile of SmGPR-1.

6.4.2 SmGPR-3 has an atypical pharmacological profile SmGPR-3 is a member of the same clade as SmGPR-2 and SmGPR-1 and is activated by dopamine and epinine (section 5.4.8).80 Other catecholamines were also able to induce a response, but these responses were weaker. SmGPR-3 is an important neuronal receptor and is likely to be involved in the control of motor activity in S. mansoni. Furthermore, the sequence homology as well as the predicted binding site differ from those of mammalian receptors. This section analyzes the pharmacological profile of SmGPR-3.

SmGPR-3 cDNA was expressed in S. cerevisiae strain YEX108 and grown in a histidine-deficient medium. This is a histidine auxotrophic strain that expresses a HIS3 reporter gene under the control of a FUS1 promoter.109 To study the effects of BA antagonists on the activity of the receptor, cells were incubated with test antagonist and agonist (dopamine, 100 μM) or vehicle (Figure 39A).80 The same concentration was used for every test antagonist (100 μM) except flupenthixol (10 μM). For the chemical structures of the compounds tested see Figure 36 and Figure 38. Dose-response curves were obtained for the most potent inhibitors (Figure 39B–F).80

The results showed that SmGPR-3 exhibited an unusual drug profile, which is not similar to that of any adrenergic or dopaminergic mammalian receptor known to date. Two of the antagonists that -6 were most effective at inhibiting SmGPR-3 activation were flupenthixol (IC50 = 3.9x10 M) and -6 haloperidol (IC50 = 1.4x10 M), which are both classical dopamine antagonists. Furthermore, -5 promethazine (IC50 = 2.8x10 ) belonged to the most potent inhibitors of SmGPR-3, which is an anti-histaminic drug. Interestingly, cyproheptadine was able to produce significant inhibition (± 70%). Cyproheptadine normally targets histaminergic and serotonin receptors. Likewise, mianserin was able to inhibit SmGPR-3 activity, which is a mixed adrenergic/serotonin/H1R antagonist. Remarkably, anti-dopaminergics like spiperone and clozapine did not result in significant inhibition or even enhanced the activity of the receptor. To test for drug-induced toxicity, the

90 experiments were repeated in medium supplemented with histidine, which results in cell growth irrespective of activation of SmGPR-2. The results revealed normal cell growth, indicating that there was no drug-induced toxicity (Figure 39A).80

Figure 39: Pharmacological profile of SmGPR-3. A) SmGPR-3 cDNA was expressed in S. cerevisiae strain YEX108 and grown in a histidine-deficient medium. The cells were treated with dopamine (100 μM) and test antagonist (100 μM or 10 μM (flupenthixol)) or vehicle (ND). An Alamar blue fluorescence assay was used to quantify receptor activation based on yeast growth (RFU, relative fluorescence units). A test for drug induced toxicity was performed (His +ve control). The test antagonists were: buspirone, BUSP; clozapine, CLZP; cyproheptadine, CPRH; flupenthixol, FLPX; haloperidol, HLRD; mianserin, MINS; promethazine, PRMZ; propranolol, PROP; spiperone, SPIP. B) Experiments were repeated with different concentrations of antagonists.80

In summary, SmGPR-3 was strongly inhibited by flupenthixol, haloperidol and promethazine. Two of these antagonists, flupenthixol and promethazine are were also able to inhibit SmGPR-2 activity (section 6.4.1). This suggest that members of the SmGPR clade, even though their ligands vary, might have similar pharmacological properties. The unusual drug profile when compared to mammalian receptors further supports the notion that SmGPR-3 can be utilized to develop schistosome-specific drugs that are aimed at disrupting worm motility within the mammalian host.

The next section describes the pharmacological profile of Sm5HTR. In addition, the pharmacological divergence between Sm5HTR and the closest human homolog is analyzed.

6.5 Pharmacological profile of serotonergic receptor Sm5HTR 6.5.1 Real-time biosensor assay to profile Sm5HTR Serotonin is a key regulator of muscle contraction in S. mansoni and serotonergic GPCR Sm5HTR was shown to be in control of larval and adult worm motility (section 5.5.3).31 For this reason, Sm5HTR is a promising drug target. Recently, a miniaturized screening assay was employed to pharmacologically profile this receptor.168 This method uses a split luciferase based biosensor that is sensitive to cellular cAMP levels to monitor the activity of Gi and Gs-coupled GPCRs. This technique named GloSensor is based upon a crucially permutated variant of firefly luciferase that contains a binding domain for cAMP from PKA. Binding of cAMP results in an enhanced luminescent signal due to a conformational change in the molecule.

In order to assess the pharmacological divergence between Sm5HTR and the closest human homolog Hs.5HT7R (30% amino acid identity),31 the GloSensor assay was used to screen a GPCR compound library for inhibitors of these GPCRs.168 HEK293 cells were transfected with Sm5HTR or Hs.5HT7R, after which they were exposed to various compounds (10 μM). This was followed by addition of serotonin (EC80 concentration), to assess inhibition of serotonin effects by the previously added compounds. The results are visualized in heat maps, which represents the pharmacological profiles of Hs.5HT7R and Sm5HTR (Figure 40). This figure shows the extent of pharmacological divergence between the schistosome and human GPCR. After removal of false positives, 23 compounds were selected as potential antagonists of Sm5HTR. Only seven of these showed inhibition at both the schistosome and human receptor.

Figure 40: Drug profile of Sm5HTR. HEK293 cells were transfected with either Sm5HTR or Hs.5HT7R and pGloSensor-22F. Subsequently, the cells were treated with a test compound (10 μM). After 30 minutes, serotonin was added (EC80 concentration). Subsequently, luminescence was measured (60 minutes). Heat maps of various ligands that represent ligand-evoked changes in cAMP levels. S. mansoni Sm.5HTR (left) and its homolog human Hs.5HT7 (middle). Each colored box depicts the fold change in luminescence after the cells were treated with a test compound and serotonin. A pseudo-color scale was used. The black boxes represent the false ‘hits’. Venn diagram displays the selectivity of compounds for either Hs.5HT7R, Sm5HTR, or both.168

Analysis of structural data from various assays provided insight into the structural selectivity between human and flatworm receptors (Figure 41).168 The ligands that exhibited preferential selectivity for Sm5HTR over the human homolog included ergot alkaloid bromocriptine. This ligand was able to cause long lasting and potent inhibition at Sm5HTR. Moreover, various potential Sm5HTR antagonists with dimethoxyisoquinoline substructures (rotudine, tetrabenazine, rotundine and tetrandrine) were found that exhibited bias and potency toward Sm5HTR over Hs.5HT7R. These promising ligands were screened against adult worms and schistosomula. The results revealed that only rotundine and bromocriptine were able to inhibit serotonin evoked hypermotility in both life-stages.

Several ligand classes exhibited selectivity for Hs.5HT7R (Figure 41; above the line).168 These included tricyclic and tetracyclic antidepressants, as well as sulfonyl derivatives. Although previous studies have shown that ligands belonging to these classes cause schistosomula hyperactivity,172 this does not necessarily mean that they are selective for S. mansoni receptors over human receptors. Similarity, all ergot alkaloids that were screened exhibited selectivity for Hs.5HT7R except bromocriptine. These ligand classes can be deprioritized when developing

93 antischistosomal drugs. Another interesting finding is that Sm5HTR showed a property that resembles irreversible inactivation. Similarly, previous studies showed that Hs.5HT7R can be irreversible bound and inactivated by specific ligands.173,174 Transient exposure of a compound may result in long lasting inhibition of worm motility, which minimalizes the required drug dosage. This finding enhances the appeal of Sm5HTR as an anthelmintic drug target.168

Figure 41: The structure-activity relationships for ligand classes against Sm5HTR and Hs.5HT7R. IC50 values for drugs that inhibit cAMP production via Sm5HTR and Hs.5HT7R are compared. Ligands showing selective inhibition of one receptor over the other cluster along the line. Hits in the red square represent ligands that show supra-μM potency at Hs.5HT7R and sub-μM potency at Sm5HTR. Tetrandrine (31), tetrabenazine (32), rotundine (37), bromocriptine (39). The colors of the hit represents to which ligand class the compound belongs. For the individual compounds, see article.168

Taken together, these results provided important insights into the extent of pharmacological divergence between Sm5HTR and the human homolog Hs.5HT7R. The pharmacological divergence may hamper the repurposing efforts for human serotonergic drugs. However, it also provides the opportunity for de novo ligand discovery if the pharmacological divergence between parasite and human GPCRs can be exploited to develop selective drugs for the control of parasitic infections. Furthermore, these results demonstrated the application of a split luciferase based real- time biosensor assay for profiling flatworm GPCRs that is compatible with high throughput screening. An advantage of this assay is that it reveals proximal receptor activity in real time since it directly reports cAMP levels. This assay proved useful for monitoring of effects of specific molecules on GPCR activity. The next section describes a follow-up study that applied the same biosensor.

6.5.2 Nuciferine is a potent Sm5HTR antagonist The first study that employed the cAMP biosensor (GloSensor, see previous section) found

Sm5HTR antagonists with dimethoxyisoquinoline substructures.168 In a follow-up study, the same cAMP biosensor assay was utilized to study the effects of five aporphine alkaloids that contain methoxyisoquinoline moieties on the signaling activity of Sm5HTR.175 This study contributes to a deeper understanding of structure-activity relationships at Sm5HTR. In total, four aporphine natural compounds were screened: D-glaucine, nuciferine, bulbocapnine and boldine (Figure 42A).175 Apomorphine was screened as well, which is a synthetic aporphine that was shown to inhibit contractility in schistosomula.176 The receptor was heterologously expressed in HEK293 cells and treated with the individual alkaloids.

The results showed that all five compounds caused a blunted response to serotonin (Figure 42B).175 After addition of forskolin, cAMP levels increased, indicating that the cells were still capable of generating cAMP. Dose-response curves were obtained, showing that the individual aporphines inhibited the cAMP generating effect of serotonin (Figure 42C).175 The most potent Sm5HTR blocker was nuciferine (IC50 = 0.24 μM), followed by D-glaucine (IC50 = 0.86 μM). Next, the effect of aporphine ligands on the motility of schistosomula was assessed. Interestingly, the most potent blockers of Sm5HTR were also the most potent inhibitors of schistosomula contraction (Table 2).175

The most potent ligands (D-glaucine, boldine and nuciferine) were screened against adult S. mansoni worms. The worms were separated into single sex cohorts and treated with one of the drugs (10 μM). Subsequently, the effect on the basal motility on females and males was measured. Nuciferine appeared to be the most effective ligand for females (Figure 43B), whereas all ligands appeared to impair male parasite mobility equally (~50%) (Figure 43A).175 Furthermore, each ligand decreased the effect of stimulation with increasing concentrations of serotonin (Figure 43C- D).175 In females, high doses of serotonin were able to restore motility after boldine and D-glaucine treatment. However, nuciferine action could not be reversed by serotonin in both males and females. This indicates that nuciferine is an effective inhibitor of serotonin evoked motility of S. mansoni worms.

Figure 42: Aporphine alkaloids inhibit Sm5HTR dependent cAMP production. A) Structures of the compounds that are tested. B) Effects of aporphines on Sm5HTR dependent cAMP production. HEK293 cells were transfected with Sm5HTR and pGloSensor-22F. Subsequently, the cells were treated with a test compound (solid circles, at every solid triangle 5 μM was added) or with a control (DMSO). After 30 minutes, the cells were treated with serotonin (0.8 μM, grey triangle). Forskolin (20 μM, open triangle) was added after stabilization of the serotonin response. C) Aporphines dose-dependently inhibit serotonin evoked cAMP production.175

Table 2: Comparison between IC50 values for aporphine ligands screened against Sm5HTR and larval schistosomules.175

Compound IC50 Sm.5HTRL (μM) IC50 schistosomula (μM) Nuciferine 0.24 ± 0.04 0.62 ± 0.22 D-glaucine 0.86 ± 0.22 3.9 ± 0.72 Boldine 1.1 ± 0.10 2.5 ± 1.4 Apomorphine 15.2 ± 3.3 24.2 ± 15.1 Bulbocapnine 15.2 ± 2.5 76.1 ± 17.8

Figure 43: Aporphine alkaloids inhibit S. mansoni worm motility. Effects of test compounds on female and male adult schistosomes. A) male and B) female schistosomes were treated with test compound (10 μM, 2h), or DMSO (control). C) . Effects of test compounds on serotonin evoked movement of C) male and D) female and male adult sworms. The data is shown as fold change over basal movement for the control cohort.175

The aporphines were more effective in inhibiting schistosomula compared to adult worms. This suggests that there may be additional mechanisms involved in adult worm muscle activity. In addition, transcription levels of GPCRs can vary between schistosome life-stages. The observed sex-specific differences may be explained by differences in drug absorption kinetics, rates of serotonin uptake, and/or Sm5HTR expression. Interestingly, recent studies showed that Sm5HTR is part of the sM-bM-sF group (Figure 8).17 The receptor is predominantly transcribed in male worms independent of pairing.22,177 Compared to pairing-inexperienced females (sF), transcription of Sm5HTR was found to be reduced by about 60% in sexually mature females (bF). The observations supports the notion that the observed sex-specific differences may be explained by differences in Sm5HTR transcription levels between males and females. Pharmacological profiling of Sm5HTR showed that ligands that contain dimethoxyisoquinoline substructures exhibited bias toward Sm5HTR over Hs.5HT7R (section 6.5.1).168 This is notable, since nuciferine contains this substructure and was the most potent inhibitor of Sm5HTR. The results in this section are also in agreement with the results of the study described in section 5.5.3. In that study, RNAi studies were performed to show that Sm5HTR is required for parasite motility.31

To conclude, the combination of findings show that nuciferine is a potent Sm5HTR antagonist and demonstrate that Sm5hTR inhibition correlates with hypomotility in schistosomula and adult worms. Future studies could further investigate methoxyisoquinoline aporphinoids as potential anthelmintic drugs. The next section describes the efficacy of promethazine against S. mansoni ex vivo and in a murine model of schistosomiasis.

6.6 Repurpose promethazine as an antischistosomal drug Developing new drugs is an expensive and time-consuming process.178 Therefore, it can be efficient to repurpose drugs or clinical candidates that are already approved. Promethazine is an antihistamine drug that can be used to prevent motion sickness and treat vomiting, nausea, and different allergic symptoms.179 In addition, it is used as a sleep aid or sedative. Various pharmacological studies screen promethazine (Figure 44), which is a first generation mammalian H1 antagonist, against schistosome receptors. For example, promethazine was able to strongly inhibit histamine-activated receptor SmGPR-2 (section 6.4.1).76 Furthermore, promethazine belonged to the most potent inhibitors of SmGPR-3 (section 6.4.2) and significant

99 changes in schistosome motility (Figure 30C) was observed when the animals were treated with the drug.78 Moreover, promethazine dose-dependently abrogated the elevated base activity of SmGAR (section 6.2).94 These findings highlight the potential of promethazine for the treatment of Schistosomiasis. Based on these findings, a recent study investigated the efficacy of promethazine against S. mansoni ex vivo and in a murine model of schistosomiasis.171

Figure 44: Structure promethazine

6.6.1 Promethazine reduces motor activity and is able to kill adult schistosomes ex vivo Adult schistosomes ex vivo were treated with various concentrations of promethazine (Figure 45).171 The viability was measured by monitoring the motor activity, changes in the tegument and mortality rate of the parasites for 72 hours. The IC50 value of promethazine was 10.29 (24 h) and 5.84 μM (72 h) (Figure 45B).171 The untreated parasites did not show any change in viability. Microscopy analysis revealed that when adult schistosomes were treated with promethazine, they showed spasmodic contractions followed by a reduction in motor activity. Specifically, the worms formed a loose knot (Figure 45D–F).171 The control group showed normal motor activity (Figure 45C). 171 Remarkably, coupled parasites were separated into individual female and male worms after administration of promethazine. Moreover, the oral sucker movements of the schistosomes were weakened. Since promethazine has anticholinergic properties, it was tested whether acetylcholine can reverse the effect of promethazine. Various lethal concentrations of the drug were administered to adult schistosomes. This was followed by various concentrations of acetylcholine. The results revealed acetylcholine was able to reverse the motility of the schistosomes, and the worms remained viable. This is suggestive of receptor competition or interaction.

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Figure 45: Effect of promethazine on S. mansoni worms. Adult schistosomes were treated with various concentrations of promethazine. The worms were monitored microscopically and after 72 h they were scored for survival. A) Structure of promethazine B) Viability of S. mansoni worms treated with promethazine or untreated worms (control). C) Control D) 6.25 µM promethazine E) 12.5 µM promethazine F) 25 µM promethazine G) 12.5 µM atropine H) 5 µM praziquantel.171

The IC50 value of promethazine in S. mansoni surpasses the criteria defined by the WHO for potential antischistosomal agents.180 The WHO describes the activity criteria as follows: S. mansoni adults 100% inhibition of motility at 5 μg per ml. Furthermore, the observed activity of promethazine is higher than those of other promising drugs, such as mefloquine, miltefosine, 181 artemether, and artesunate. However, the IC50 value of PZQ is 1.26 μM, which is significantly lower than that of promethazine.182,183

6.6.2 Promethazine reduced worm burden in juvenile and adult schistosomes The drug that is currently used to treat schistosomiasis, PZQ, is inactive against juvenile schistosomes.8 Possibly, this explains the low cure rate in high transmission risk areas, since patients are likely to harbor both juvenile and adult schistosomes.184,185 Therefore, it is important that a new drug is able to target schistosomes in various life stages. To test whether promethazine is effective against schistosomes in various life stages, promethazine (5 x 100 mg/kg) was administered to mice that were infected with S. mansoni. The results revealed that the worm burden reduced in juvenile (prepatent infection) as well as in adult (patent infection) schistosomes (P < 0.05 to 0.0001) (Figure 46).171 In juvenile schistosomes a total worm burden reduction of 44.7%

101 was observed. After administration of promethazine to both male and female adult schistosomes, a reduction of 94.1% was observed. To compare, praziquantel reduces >90% of the total worm burden.181,186

These results show that oral administration of promethazine is less effective against juvenile S. mansoni in comparison to adult schistosomes. This is also the case for praziquantel and previously tested promising compounds nerolidol and spironolactone.187–189 The WHO states that compounds that result in a total worm burden reduction of 80% after five doses (100 mg/kg) are highly active compounds. This means that according to WHO promethazine can be described as highly active. Since schistosomes do not multiply in the mammalian host (section 1.1), a reduced worm burden often results in reduced pathology.190

Figure 46: Effect of promethazine on worm burden. Mice that were infected with juvenile (prepatent infection) or adult schistosomes (patent infection) were treated with promethazine (100 mg/kg) for 5 days. On day 56 post infection, the worm burdens were analyzed by sex. Worm burden reduction; WBR.171

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6.6.3 Promethazine reduced the egg burden in various life-stages of S. mansoni As discussed in section 1.1, schistosome eggs play a fundamental role in schistosome life cycle and in the induction of pathogenesis.22 Therefore, a drug that can reduce egg production has a high chance of being effective. To test whether promethazine has an effect on egg burden, samples of intestinal tissue were investigated (oogram). The Kato-Katz method was utilized to measure the number of eggs in fecal samples. The results showed that promethazine significantly reduced the egg burden in various developmental stages of S. mansoni, predominantly in patent infection (Figure 47).171 The number of immature eggs was reduced by 95.3% (oogram, P 0.0001) in the adult infection model. Likewise, analysis of fecal samples showed a reduction in eggs of 96.1% (P 0.0001) (Figure 47B).171 In contrast, the number of eggs was reduced by 37.2% (Figure 47A, P0.05) and 54.2% (Figure 47B, P0.01) in the juvenile infection model.171 These results confirm that promethazine is less effective against juvenile S. mansoni compared to the adult life stage. This highlights the need to invent drugs with new mechanism of action that are active against mature and immature S. mansoni.

Figure 47: Egg burden reduction effect of promethazine. Mice that were infected with juvenile (prepatent infection) or adult schistosomes (patent infection) were treated with promethazine (100 mg/kg) for 5 days. On day 56 post infection, eggs were counted in the feces (Kato-Katz) and the intestine (oogram). Egg burden reduction; EBR.171

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6.6.4 Promethazine is likely to act on cholinergic system of S. mansoni The tegument is the major interface between the external environment and the schistosome. It is renewed every six hours and it plays an crucial role in the protection against the immune system of the host.191 Therefore, a growing number of studies that focus on the development of antischistosomal drugs have been setting the tegument as a target.192,193 Although the mechanism of action of promethazine on S. mansoni is unclear, evidence suggest that it may target the tegument. Microscopic studies revealed that schistosomes that were treated with promethazine displayed altered morphology of their surface.171 Specifically, the tegument was ruptured along the entire dorsal body surface. This effect was concentration-dependent. In contrast, the tegument of the control group remained intact. A possible partial explanation for the effect of promethazine on the teguments of adult schistosomes is that promethazine is inherently lipophilic (Figure 44). Therefore, this compound is able to easily cross the tegument and access its target(s) in the schistosomes.

Promethazine may act on the neuromuscular system of S. mansoni. Administration of the drug promethazine results in concentration-dependent changes in the motility of schistosomes and acetylcholine could reverse this effect. These observations may support the hypothesis that this compound acts on the neuromuscular system of S. mansoni. Previous studies showed that there are various putative cholinergic receptors in S. mansoni.37,194,195 Recently, a functional acetylcholine GPCR was described (SmGAR).196 This study provided evidence that this receptor is crucial for proper motor control in S. mansoni larvae (section 5.2). Moreover, it was shown that promethazine has inverse agonist activity towards SmGAR, which results in significant lower basal activity of the receptor (section 6.2). These results suggest that promethazine acts on the cholinergic system of schistosomes. The results also are in concordance with previous studies that showed that cholinergic receptors in S. mansoni inhibit neuromuscular functions.195

This study does not consider other promethazine targets in S. mansoni. However, other studies have shown that promethazine exhibits inhibitory effect on various schistosome receptors. These receptors include, SmGPR-2 and SmGPR-3.76,80 SmGPR-3 expression was detected in the peripheral innervation of the body wall muscles and suckers. If promethazine exerts an effect on SmGPR-3, this may explain the weakening of the oral sucker movements of the schistosomes and

104 the separation of schistosome pairs into individual female and male worms after administration of promethazine. Similarly, weak expression of SmGPR-2 was detected in the ventral and oral suckers.76 This receptor was shown to selectively respond to histamine and may modulate neuromuscular circuits. Furthermore, Strong SmGPR-1 expression was found in the musculature of the suckers.77 Even though this receptor is not yet pharmacologically profiled, it is likely that promethazine also inhibits this receptor since this receptor is a member of the same clade as SmGPR-2 and SmGPR-3. Moreover, SmGPR-1 expression was detected in the subtegumental musculature and SmGPR-2 is expressed in the subtegumental nerve plexus. Since the tegument of schistosomes was ruptured along the entire dorsal body surface after administration of promethazine, it is suggested that promethazine reaches its molecular target in this area. Promethazine is less effective against juvenile S. mansoni compared to the adult life-stage. This may be explained by differences in GPCR expression levels between various life-stages. For example, SmGPR-1 and SmGPR-2 are upregulated in young larval (schistosomula) life-stages compared to the adult life-stage (section 4.2).76,77 Taken together, these findings indicate that promethazine may exert its antischistosomal properties through various receptors.

Although promethazine shows promising results as an antischistosomal drug, there are some disadvantages.171 The effective daily dose of 100 mg/kg used in the mouse model is translated to 8 mg/kg for humans.171,197 This dose is more than 3-fold higher than the recommended dose, which would result in sedative effects and delirium in humans.197,198 In conclusion, although promethazine could possibly be repositioned as an antischistosomal agent, there are still limitations to overcome in clinical practice. Future work could focus on unravelling the mechanism of action of promethazine in S. mansoni. This may reveal that it targets GPCRs, which could lead to the development of antischistosomal drugs using an GPCR structure-based drug design.199,200 Similarly, more (structural) information on the target helps in the design of effective phenothiazine derivatives against Schistosomiasis.

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6.7 Summary This chapter analyzed the pharmacological profiles of deorphanized receptors. In addition, the extent of pharmacological divergence between Sm5HTR and Hs.5HT7R was analyzed. Lastly, the possibility of repositioning promethazine as an antischistosomal drug was discussed. Interestingly, the pharmacological profiles of schistosome receptors showed to be substantially different from those of receptors in the mammalian host. For example, the pharmacological profile of SmGluR differs from that of mammalian mGluRs.67 Only one of the classical glutamate antagonists was able to produce a modest inhibition of SmGluR activity and none of the classical agonists activated the receptor. Similarly, promethazine was the only histaminergic that produced a significant inhibition of SmGPR-2 activity, which is atypical for a histamine receptor.76 The pharmacological profile of SmGPR-3 shared similarities with the profile of SmGPR-2.78 Since these receptors belong to the same clade, it is suggested that members of this clade have similar pharmacological properties even though their ligands differ. Future studies could pharmacologically profile SmGPR-1 to investigate whether this receptor shares these pharmacological properties as well. The pharmacological profile of SmD2 also proved to be atypical. Even though the receptor responded to some classical dopamine agonists, their relative affinities differed from mammalian D1 or D2 profiles.66 Most notable, a classical mammalian dopamine agonist acted as an antagonist with high potency on SmD2. The unusual drug profiles of schistosome receptors compared to mammalian receptors suggest that selective antiparasitic drugs can be developed. This is supported by a study that developed a method that evidences the pharmacological divergence of a schistosome receptor from the closest human homolog.168,175 The divergence in pharmacological selectivity between the Sm5HTR and the human homolog supports the feasibility of optimizing schistosome selective pharmacophores.

Taken together, these results support the rational design and development of antischistosomal agents that target GPCRs. Future studies focusing on a variety of schistosome GPCRs could apply the same biosensor that was employed for screening different ligands against Sm5HTR. This may reveal additional ligand classes that exhibit selectivity and potency towards parasite receptors.

A study demonstrated potent schistosomicidal activity of promethazine, which is a H1 receptor antagonist developed for targeting mammalian receptors.171 However, the repurposing of this drug

106 is limited in clinical practice by large drug doses and unwanted side effects. Future studies may focus on designing phenothiazine derivatives that are effective against Schistosomiasis. Promethazine may target acetylcholine receptor SmGAR in S. mansoni, since this compound acts as an inverse agonists on this receptor and acetylcholine was seen to reverse the parasite’s motility.94,171 Atropine was shown to be the most potent inverse agonist that acts on this receptor.94 These results showed that inverse agonists have potential for GPCR and anthelmintic drug discovery.

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7. Discussion

7.1 GPCR families The aim of this paper was to explore the possibility of schistosome GPCRs as anthelmintic targets. This study analyzed the identification and classification of putative schistosome GPCRs at the family level and investigated the expression of schistosome GPCRs in various life-cycle stages. Based on whole genome sequencing data, 115 putative GPCRs were found with a minimum of three predicted TMs.17 105 of these GPCRs had more than four TMs and were analyzed phylogenetically. In total, 67 seven-transmembrane receptors were identified. The results revealed that all major families were represented, which indicates that GPCR signaling is important in S. mansoni. The majority of the GPCRs that were identified belong to the α or β Rhodopsin subgroup. The amine grouping is largest for S. mansoni GPCRs. Interestingly, biogenic amines have been shown to be widely distributed in the nervous system of schistosomes and they play prominent roles as modulators of neuromuscular function and motility.28 Since locomotion plays a crucial role in the survival of the parasite, GPCRs that are targeted by biogenic amines are potential targets for antischistosomal drugs.31

Another GPCR family that is of interest is the new and highly-diverged Platyhelminth Rhodopsin Orphan Family (PROF).15 No readily identifiably similarity to non-flatworm GPCRs was found, which indicates that this is a platyhelminth specific GPCR family. Moreover, receptors that are housed in this family may act as targets for selective anthelmintics. Natural ligands of this GPCR are likely to be flatworm-specific peptides.17 Determining these ligands and receptor function will prove challenging due to the fact that the family is highly diverged. However, patterns of GPCR expression during the course of the development of S. mansoni can provide insight into potential functions. For example, Smp_041880 is a putative PROF1 receptor. Analysis of life-stage specific expression data showed that Smp_041880 is equally expressed in juvenile (schistosomula) and adult worms, while the expression level in cercaria is significantly lower.47 In contrast, the majority of the PROF complements followed a general pattern of increased expression in the juvenile schistosomula life stage. Tissue-specific transcriptome analyses showed that Smp_041880 is grouped in the bF group and is predominantly described in the ovary of female schistosomes.17 This may indicate that this receptor plays an important role in in the egg-laying stages. This finding

108 and the receptor’s platyhelminth specific family indicate that drugs that target this receptor may be parasite-specific and potentially disrupt the reproduction process of S. mansoni.

The majority of the putative PROF1 receptors are upregulated during the parasitic stages of S. mansoni. This indicates that drugs that target PROF1 receptors are effective against both juvenile and adult schistosomes,47 increasing the chance that the parasite is eliminated from the host. Since these receptors appear to be restricted to, and expanded in, the phylum Platyhelminthes, further research into this family may reveal important functional innovations that are specific to this phylum. Future studies must focus on identifying the ligands and functions of PROF receptors. These studies could use various strategies that have been described for flatworm parasites. These include RNA interference as a genetics strategy to silence expression of PROF genes. There are multiple protocols available for producing RNAi in various life-cycle stages of S. mansoni.38,201– 207 This method can be combined with imaging assays in order to monitor the behavior of the worms and to quantify movement in culture.208 Furthermore, if the heterologous expression of PROF receptors can be achieved, they could be screened with the growing canon of peptide ligands from Platyhelminthes and other genera using a receptor activation assay.44,45,209 In addition, spatial expression patterns could provide insight into potential functions.52

On a critical note, there is no set definition for lineage specificity in the literature on Platyhelminthes GPCRs. For instance, previous studies describing PROF1, RhoL/R and Srfa/b/c receptors employed different criteria and methods.15,49 A recent study that profiled GPCRs of F. hepatica noted that BLASTp searches of a lot of flatworm GPCRs reported as taxonomically restricted against non-flatworm sequence datasets still returned high scoring matches.52 For example, this is the case for PROF1 receptors in S. mansoni and S. mediterranea described in 2011, potentially resulting in exclusion of a part of these receptors. However, a more recent and updated phylogenetic analysis described a highly supported PROF clade.17 Moreover, the receptors included in this study are linked to a gene model validated by previously conducted RNA-seq experiments.37 Nonetheless, a more stringent and set definition of taxonomically restricted GPCRs would benefit research on orphan GPCRs.

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The described phylogenetic analysis of GPCRs included 105 receptors with more than 4 TMs. However, it is unclear whether the putative GPCRs that contain less than seven TMs act as functional GPCRs. There is limited evidence that GPCRs that contain fewer than seven TMs act as functional receptors in the aspects of receptor expression, signaling, desensitization and internalization.210 Moreover, all functionally characterized GPCRs in S. mansoni contain 7 TMs.17 However, the assumption that all GPCRs should contain 7 TM regions may be incorrect.211

Further research should be undertaken to characterize the putative GPCRs that remain to be deorphanized. These include amine and PROF1 receptors (Rhodopsin Family receptors), the receptors that were classified as Glutamate-like and Adhesion-like receptors and the Frizzled and Smoothend-like sequences. The description of the S. mansoni GPCR complements provides important sequence and functional leads to deepen the understanding of schistosome receptor biology. Moreover, the data provide potential candidate receptors for functional studies, invigorating the exploitation of GPCRs for the discovery of antischistosomal drugs.

7.2 GPCRs in different tissues and life-stages One method that provides new insights into potential GPCR functions is a tissue-specific transcriptome analysis. A recent study analyzed the tissue-specific and pairing dependent GPCR expression in S. mansoni. The results revealed that GPCRs are expressed in whole worms as well as gonads and largely in a pairing-dependent manner.17 The GPCRs that have a non-gonad- preferential, pairing-dependent transcript profile may play a role in the interaction between male and female schistosomes.17 These GPCRs are potential antischistosomal drug targets since the male-female interaction is crucial for the induction of germ-cell differentiation. Potential functions of these GPCRs may include partner attraction, locomotion and clasping and pairing processes. A part of the analyzed GPCRs had a gonad-preferential transcript profile.17 These GPCRs may contribute to embryogenesis, vitellogenesis and/or gametogenesis. Since schistosome eggs play a critical role in schistosome life-cycle and in the induction of pathogenesis, identifying receptors that mediate egg production and gonad development will help in the identification of drug targets in S. mansoni.

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The majority of the transcripts of GPCRs that were analyzed occurred in a higher abundance in bM, sM and sF compared to bF.17 This general pattern of transcriptional activity has also been observed for Sm_npps.212 This indicates that some GPCR may act as Sm_npp targets. This hypothesis is supported by a recent study that identified certain neuropeptides as potential ligands for GPCRs.85 Furthermore, this general pattern of transcriptional activity may be indirect evidence for the hypothesis that female schistosomes ‘hand over’ the responsibility for maintaining the majority of the neuronal circuits that involve neuropeptide signaling to the males after they have paired.212

Pairing-inexperienced males (sM) already possess complete seminal vesicles and testes with differentiated sperms and are not morphologically different from pairing-experienced males (bM).60,61 Nonetheless, it has been known that sM and bM gene expression differ as a result of pairing.22,62 Previous studies have assumed that genes, including GPCR genes, that are differentially transcribed in the male after pairing are associated with enhanced muscular power in order to transport the paired female.17 However, a recent study showed that the male schistosome’s effort is not restricted to this function and nutritional support.212 Instead, a more complex pairing scenario emerged in which both male and female schistosomes live in a reciprocal sender-recipient relationship. This relationship does not just affect the gonads of male and female schistosomes but may involve neuronal processes that include neuropeptides and GPCR signaling. In addition, transforming growth factor β-dependent signaling and tactile stimuli may be involved. These different signaling system mediate the communication between both genders. Evidence suggest that the management of some neuronal processes shifts to the male schistosome upon pairing. Unravelling differentiation programs in male and female schistosomes may reveal potential antischistosomal drug targets. For example, targeting GPCRs that are involved in communication between male and female schistosomes may result in disruption of the pairing process and thereby eliminating the parasite from the host.

The tissue-specific transcriptome GPCR analysis was limited by data provided by a comparative RNA-seq analysis that used an organ isolation method.17,22,65 A few deorphanized GPCRs were absent from the transcriptome data. These included SmGPR-1, SmGPR-2 and SmGPR-3, which were shown to be expressed in adult schistosomes.76,79,80 Although the organ isolation method that

111 was used claimed to surmount detection limits of other methods such as immunolocalization and in situ-hybridization,65 these SmGPR-like receptors were absent due to transcript levels below threshold. This indicates that an increase in sensitivity regarding low abundant proteins and transcript would help detect even more GPCRs. In addition, future studies on organ-isolation methods could focus on the enrichment of tissues other than the gonads, such as the intestine and the vitelline lobes.65 This would help connect GPCRs to specific biological functions in these tissues.

The data provided by the comparative RNA-seq analysis also revealed that approximately 60% of the GPCR genes are expressed in adult S. mansoni,1,26 which indicates that GPCRs are low abundantly expressed in adult schistosomes in comparison to different life-cycle stages of S. mansoni. Therefore, this paper analyzed the schistosome GPCR expression in various life-cycle stages. Patterns of GPCR expressing during the course of the development of S. mansoni provides imports insights into potential functions. One interesting pattern that emerged from transcriptome sequencing and microarray analyses of S. mansoni is that some neuroreceptors appear to be upregulated in schistosomula.37 For example, biogenic amine receptors SmGPR-1 and SmGPR-2 and glutamate receptor SmGluR are upregulated during the development of early schistosomula.67,76,77 This is followed by down-regulation during the transformation into adult schistosomes. Furthermore, dopamine receptor SmD2 appears to be upregulated in schistosomula and cercariae compared to adult worms, although this is not confirmed at mRNA level yet.66 The expression of acetylcholine receptor SmGAR is also predicted to be highly upregulated during the early larval stages of S. mansoni.37,94 Furthermore, PROF genes appear to be up-regulated in schistosomula as well.

Taken together, these results indicate that GPCRs in adult schistosomes are generally low- abundantly expressed compared to schistosomula. The gene expression pattern of neurotransmitter receptors suggest that the larval nervous system undergoes broad changes during its development. These upregulated GPCRs may be particularly important during the active migration of larvae in the bloodstream of the host. Identification of these GPCRs helps unravelling critical pathways during this migration. RNAi can be performed in the larval stage tor the functional validation of these GPCRs. For example, the results of an RNAi study indicated that SmGAR plays a role in

112 larval motility and its activity results in the movement of young larvae.94 These results further support the hypothesis that SmGAR is involved in early parasite migration.

The GPCRs that are abundantly expressed in schistosomula are interesting potential drug targets because it is desired that the next antischistosomal drug is effective against this life-stage. Especially since the currently available drug to treat Schistosomiasis, PZQ, is not effective against juvenile schistosomes.8 However, the next antischistosomal drug must be effective against adult worms as well. Fortunately, the receptors that are upregulated in schistosomula are also expressed in adult schistosomes. For example, the SmGPR-like receptors are expressed in the nervous system of adult schistosomes.76,79,80 Moreover, SmD2 was shown to be predominantly expressed in the somatic muscles and in a lower amount in the muscular lining of the caecum of adult worms.66

7.3 Deorphanized GPCRs Phylogenetic analysis of schistosome GPCRs is likely to help in deorphanizing GPCRs. In mammalian phylogenetic analyses, the chemical nature of endogenous agonists significantly overlap with the clustering of GPCRs.34,35,213–215 Similarly, assignment of GPCRs to specific functional groups may help tremendously in their deorphanization. Despite recent advances, however, most schistosome GPCRs have yet to be characterized by means of cloning and functional expression assays. Thus far, seven GPCRs have been deorphanized. This paper has investigated the characterization of these receptors.

When comparing the deorphanized receptors, similar features can be observed. First, several schistosome GPCRs exhibit constitutive activity in the absence of ligand. These include SmGPR- 2, a receptor that is further activated by histamine, and SmGAR, which could be activated above its elevated baseline by acetylcholine.76,94 SmGPR-3 exhibited partial constitutive activity and could be further activated by catecholamines.78 Similarly, dopamine receptor SmD2 had some constitutive activity in the absence of a ligand.66 These results suggest that some schistosome GPCRs have a propensity towards spontaneous activation. This propensity has been described for many other GPCRs.111 In addition, evidence suggest that constitutively active GPCRs are of biological relevance.111,216 Constitutive activation may play a role in modulating the basal activity of GPCRs in vivo and, as a result, the magnitude of agonist response. Moreover, endogenous inverse agonists may target GPCRs that exhibit constitutive activity, resulting in lowering of the

113 basal activity of the receptor.169 This is another mechanism by which agonist responses can be fine- tuned.

However, the constitutive activity observed for a GPCR is simply manipulated in vitro by overexpressing G proteins.100 Therefore, it may be the case that the observed constitutive activity is a function of heterologous expression. For many GPCRs, mutational changes made in specific regions can result in elevated basal activity.217,218 A region called the ‘ionic lock’ of rhodopsin- like receptors is shown to be important for shifting between inactive and active receptor states.94 Remarkably, a study showed that there is likely an association between the non-conservative amino acid substitutions in the ‘ionic lock’ region of SmGAR and the receptor’s constitutive activity.94 Interestingly, most invertebrate GAR-like sequences carry non-conservative amino acid substitutions in the ‘ionic lock’ region. This may point towards a difference in the mechanism of conformational activation between vertebrate and invertebrate GPCRs. This difference may be exploited to develop parasite-selective drugs. Future studies could investigate whether other GAR- like receptors exhibit constitutive activity.

In contrast, structural analysis of SmGPR-2 showed that most amino acids that are implicated in the normal constraints on the activation of GPCRs are conserved in SmGPR-2.152 The authors noted that the D3.32N substitution might contribute to the destabilization of the inactive conformation. However, the paper does not compare the constitutive activity of SmGPR-2 to SmGPR-3. SmGPR- 3 is the only receptor in the SmGPR-like clade where D3.32 is conserved, and this receptor also exhibits (partial) constitutive activity.80 The conserved aspartate may contribute to the lower level of constitutive activity compared to SmGPR-2. However, it is likely that multiple residues are involved in the observed constitutive activity of both receptors.

Future studies should determine whether the high basal activities of some schistosome GPCRs is relevant in the parasite in vivo or a function of expression in heterologous environments. Furthermore, future investigations should use inverse agonists to characterize the pharmacology of constitutive active schistosome GPCRs.169 In recent years, there has been an increasing interest in the therapeutic benefits of inverse agonists.149,169 Research has shown that compounds that are known as competitive antagonists, can act as inverse agonists when they are tested on receptors

114 that are constitutively active. The only study that has employed this approach is the study that investigated SmGAR.94 The results of this study showed that promethazine and atropine each dose- dependently abrogated the elevated base activity of SmGAR. Since the inhibition occurred without the presence of acetylcholine, the compounds acted as inverse agonists. Although the study on SmGPR-2 did not employ an active approach to investigate the effect of inverse agonist on the receptor, the addition of promethazine at 10-4 M inhibited all (constitutive) receptor activity.76 With regard to SmD2 and SmGPR-3, the data obtained in the pharmacological studies were normalized to the control containing dopamine but no antagonist.66,80 Therefore, no inverse agonism could be observed.

The results of these studies show that constitutive activity can be observed in schistosome GPCRs when expressed in heterologous systems. Therefore, it is important to use proper controls in future deorphanization studies since high basal activity of schistosome receptors could lead to false positives when looking for an agonist.28

Another similar feature that can be observed when comparing the functionalization studies of schistosome GPCRs is the unusual signaling mechanisms. The first receptor that appears to signal through an unusual pathway is SmGluR. Glutamate activated the receptor and stimulated the cAMP production dose-dependently.67 This suggests that the receptor is coupled to a Gs type protein, which stimulates adenylate cyclase. In contrast, mammalian mGluRs usually couple to Gi/o and decrease the CAMP level or bind to Gq/11 and increase intracellular Ca2+. Recent heterologous expression studies, however, have shown that mGluRs can signal through other mechanisms as well. For example, activation of mGluR1a results in an increase in cAMP levels by coupling directly with a Gs protein.219 The second receptor that signals through a different pathway compared to its human homolog is SmD2. The receptor is most likely Gs-coupled and signals through stimulation of cAMP levels system.66 No detectable change in Ca2+ levels was measured. In contrast, mammalian D2 dopaminergic receptors signal by stimulating the Ca2+/phosphoinositol pathway or by decreasing cAMP levels.115 It is unknown whether these unusual mechanisms are functions of the heterologous expression environment or if they also occur in vivo.

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Some schistosome GPCRs signal through a typical pathway. For example, Sm5HTR signals through in an elevation of cAMP, which is consistent with a 5HT7 signaling mechanism.31 Histamine activation of SmGPR-1 triggered mobilization of intracellular calcium but not cAMP, indicates that receptor may be coupled to Gq/11 proteins and transduces through the inositol phosphate pathway. This receptor shared the same level of sequence homology (±30%) with all major types of amine GPCRs, including H1, H2, H3 and H4. Therefore, the signaling mechanism of SmGPR-1 cannot be compared to a (mammalian) homolog. Even though there is no clear overall homology with any histamine receptor class, SmGPR-1 shares some structural characteristics with H1 receptors. This type also signals preferentially by linking to Gq/11 proteins.

Members of the same clade as SmGPR-1 are histamine-responsive receptor SmGPR-2 and dopamine-responsive SmGPR-3. These receptors are likely coupled to Gq proteins since chimeras of GPA1 and human Gαq yielded significant receptor activity in a yeast functional assay, whereas the use of other chimeras (Gαo, Gαi2, Gα12, Gαs) yielded in lower or no receptor activity.76,80 However, no signaling assay was used that measured changes in cAMP or Ca2+ following receptor activation. Instead, a GPCR assays based on a HIS3 reporter gene expressed in yeast was used.99 A similar approach was employed for the functional analysis of acetylcholine receptor SmGAR.94 The receptor is likely coupled to Gαi proteins since the chimera of GPA1 and human Gαi yielded receptor activity. However, no yeast strains carrying Gαo, and Gα12 were tested. Muscarinic acetylcholine receptors (mAChRs) typically cause a change in intracellular cAMP or calcium levels.

There is abundant room for further progress in determining schistosome GPCR signaling pathways. Future studies could use different GPCR assays since GPCR signaling is not limited by changes in intracellular Ca2+ and cAMP. Assay that can measure the activity of more diverse GPCRs include assays that can measure protein phosphorylation events, like ERK phosphorylation.220 Furthermore, β-arrestin recruitment is a distinct intracellular signaling pathway, and ligand-GPCR interactions may be (completely) biased towards activating the β-arrestin pathway over classical GPCR mechanisms.221–225 Assays that use the interaction between β-arrestin and the receptor could potentially be used to further functionally characterize schistosome GPCRs.226 In addition, screening the recruitment of β-arrestin may help the identification of endogenous ligands for

116 orphan GPCRs.225 These two types of assays do not require interaction with a G protein. Moreover, these approaches have not yet been employed in helminths.28 Another method that could be used is the recently reported nanoluciferase-based complementation assay.227 This technique can be used to profile compounds that promote an interaction between a receptor and a single G protein. In addition, it can be used for high-throughput screening. Another advantage is that it can be applied to detect arrestin recruitment. Therefore, biased agonism can be detected by measuring G protein recruitment and β-arrestment recruitment using the same technique.

Discovering a natural ligands of a receptor and matching a receptor with the G protein(s) they activate in vitro is the first step towards the elucidation of the biological function. However, it is challenging to use this information to assign a biological role to the receptor in the context of the whole worm.28 As previously mentioned, RNAi can be used to determine whether a receptor produces a motor phenotype in RNAi-suppressed S. mansoni. This method was used to study SmGAR and Sm5HTR. The results suggested that SmGAR is required for proper motor control and its activity likely stimulates motility in S. mansoni larvae. The same hypoactive phenotype was observed for animals that were treated with Sm5HTR siRNA. Immunolocalization can be used to investigate the tissue distribution of a receptor. The tissue distribution of a receptor can provide important leads for unravelling a receptors biological role. Immunolocalization studies of Sm5HTR revealed that the receptor is expressed in nerve tissue. This suggests that serotonin acts through this receptor to stimulate neuromuscular and/or interneuronal signaling and by doing so stimulates movement indirectly. It is unclear how SmGAR influences worm motility. SmGAR and Sm5HTR are the only parasitic flatworm GPCRs that were demonstrated to be functionally essential.228 Moreover, their importance for worm movement identifies them as interesting anthelmintic targets.

Although RNAi can be a useful technique, there are some disadvantages to overcome. For example, the knockdown efficiency can vary greatly depending on the target.8,205,229 Furthermore, off-target effects can be a problem and knockdown is often partial.230 Therefore, phenotypes may be masked when a reasonable expression level remains. Moreover, defining RNAi phenotypes other than paralysis, death and egg production remains challenging. RNAi can be performed in vivo via dsRNA introduced (for example by electroporation) in schistosomula, after which they are introduced into mice.202 However, gene suppression may not last long enough for detecting a

117 phenotype in vivo and the number of parasites that can be recovered from infected mice is often small.231 Future studies may perform gene silencing by injecting a mouse with parasite specific siRNAs or morpholinos.232–234 These studies could apply these methods to the SmGPR-like receptors, SmGluR or SmD2 to gain insight into their biological roles.

Although siRNA has not yet been performed on these receptors, immunolocalization studies have provided functional leads. Dopamine receptor SmD2 is expressed in the somatic muscles, whereas dopamine receptor SmGPR-3 is associated with neuronal elements in the body wall.66,80 Both receptors are likely involved in dopaminergic mechanisms that control worm motility. SmGPR-3 likely mediates an indirect neuronal pathway, while SmD2 acts directly on the body wall muscle. Since exogenous applied dopamine results in paralysis of larvae, it was predicted that antagonists of SmGPR-3 results in hyperactivity.106 However, promethazine and flupenthixol induced inhibition of motor activity. This finding further indicates that multiple dopaminergic pathways play a role in motor activity. Furthermore, promethazine and flupenthixol have been shown to interact with various schistosome receptors, indicating that these two compounds exert their effects through multiple receptors. For this reason, RNAi studies should be performed to discover whether SmGPR-3 and SmD2 are essential for parasite motility.

Immunolocalization analysis using a specific antibody of SmGluR revealed that the receptor is widely expressed in the nervous system of S. mansoni. Moreover, SmGluR is associated with the epithelium of the reproductive tract or the oocytes, similar to what has been observed for certain mammalian mGluRs.92,93 Future studies could investigate whether this receptor could be a drug target to reduce or eliminate ova, which is a major cause of pathology in schistosomiasis. The two histamine receptors that have been identified in S. mansoni are SmGPR-1 and SmGPR-2.76,79 SmGPR-1 is located in the body wall musculature of the parasite, whereas SmGPR-2 is expressed near histamine-containing neurons and is enriched in the neurons of the subtegumental nerve plexus. The location of SmGPR-1 and SmGPR-2 suggest that they could contribute to schistosome motility, either by direct activation or via regulation of neuromuscular circuits. These two receptors potentially play a role in host-parasite interactions by responding to endogenous (host-derived) histamine.235 Immunolocalization studies can be expanded by using selective antibodies individually as well as in combination. This way, the spatial relationships between different

118 aminergic pathways can be examined.28

Taken together, the results of these functional studies showed that schistosome GPCRs mediate many important biological functions, including worm motility and potentially oogenesis. In addition, the receptors that are expressed in the nervous system of S. mansoni are appealing drug targets because the parasite relies on its nervous systems to coordinate essential behavior for the survival in the mammalian host.

7.4 Pharmacology of schistosome GPCRs Although the druggability potential of mammalian GPCRs has been established,236–238 much less is known about the pharmacology of schistosome GPCRs. Therefore, this paper analyzed the pharmacological profiles of schistosome GPCRs. Interestingly, the pharmacological profiles of schistosome GPCRs showed to be substantially different from those of GPCRs in the mammalian host. Dopamine receptor SmD2 phylogenetically clusters with D2 sequences, but its pharmacological profile does not resemble any mammalian dopaminergic receptor. Most notable, apomorphine, a classical mammalian dopamine agonist, acts as an antagonist with high potency at heterologously expressed SmD2.66 SmGPR-3 is a member of a schistosome specific biogenic amine GPCR clade and is most potently activated by dopamine.80 When treated with classical biogenic amine antagonists, SmGPR-3 displayed atypical pharmacology. Most surprisingly, mammalian D2 antagonist spiperone behaved like an agonist at heterologously expressed SmGPR-3. SmGPR-3 has not yet been treated with apomorphine, whereas a study showed that spiperone inhibited SmD2 activity.

Remarkably, two of the most potent inhibitors of SmGPR-3, flupenthixol and promethazine, were the most potent inhibitors of SmGPR-2 as well.76 This indicates that SmGPR-like receptors may share pharmacological properties despite differences in their natural (amine) ligands. Future studies should pharmacologically profile SmGPR-1 to investigate whether this receptor also shares these pharmacological properties. Histamine activated receptor SmGPR-2 has atypical pharmacology as well, since the only histaminergic that produced a significant inhibition of SmGPR-2 was promethazine.76

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SmGluR is activated by glutamate, but the its pharmacological profile differs from that of mammalian mGluRs. Only one of the classical glutamate antagonists (LY341495) produced a modest inhibition of SmGluR activity and none of the classical agonists activated the receptor. Another study investigated the LBD of this receptor and described the LBD as atypical, with only one putative glutamate-interacting residue.15 The authors predicted that this receptor may bind other amino acid-derived ligands or has atypical pharmacology. However, the study that functionally characterized SmGluR stated that several residues implicated in glutamate binding were identified, including, Asp 208, Tyr 236, Ser 165 and Arg 323.67,90 The reason for the discrepancy is unclear. Homology modeling of the glutamate receptor may give insight into the binding site of glutamate and the structural organization of the receptor. This has been done for SmGPR-3,80 which is possible due to high-resolution crystal structures for biogenic amine and mammalian rhodopsin receptors.153,154 These structures can be utilized as a template to generate a model of schistosome receptors.

Taken together, these studies suggest that schistosome GPCRs have different pharmacology from mammalian GPCRs. This indicates the enormous potential of schistosome GPCRs as targets for schistosome-specific drugs. This is supported by a study that developed a method that evidences the pharmacological divergence of a schistosome receptor from the closest human homolog.168,175 The divergence in pharmacological selectivity between the Sm5HTR and the human homolog supports the feasibility of optimizing schistosome selective pharmacophores. The technology that was used allows for chemical library screening and real-time monitoring of the activity of GPCRs and is competent to resolve different types of GPCR modulators (allosteric modulators, partial, inverse agonists, full). Other pharmacological studies tested GPCRs with only a small selection of ligands that were selected around inferred agonist specificity. This method may detect only certain pharmacological properties. Schistosome-specific drug development efforts can greatly benefit from structure-activity profiling since it can provide a deeper understanding of pharmacophores that are selective for specific receptors.239

The pharmacological divergence between Sm5HTR and its mammalian homolog showed that repositioning efforts for existing mammalian serotonergic drugs may be limited.168 A study that investigated the potential of repurposing promethazine as an antischistosomal drug showed that

120 even though promethazine was able to kill schistosomes ex vivo, the effective daily dose translated for humans would be too high. This supports the notion that repurposing efforts for drugs designed for human usage may be limited. However, it is likely that promethazine targets various GPCRs, including SmGAR, indicating that drugs that target schistosome GPCRs have potential to kill schistosomes and reduce worm burden. Future studies could investigate promethazine analogs as antischistosomal agents.

7.5 Conclusion and outlook To conclude, this paper showed that all major GPCR subfamilies are represented in the genome of S. mansoni. Moreover, previous studies have identified several S. mansoni receptor that are members of the flatworm specific GPCR family (PROF1). These are appealing potential candidates for specific anthelmintic drug targets. However, their natural ligands remain to be identified. Description of the GPCRome of S. mansoni allows for employing a GPCR target-based screening approach for antischistosomal drug discovery. Another finding that emerged from this study is that GPCRs are expressed in different schistosome tissues. Most GPCRs have a non-gonad-preferential, pairing-dependent transcript profile. Drug that target these receptors may cause disruption of non- gonad, pairing-dependent processes in S. mansoni. GPCRs that are expressed in the nervous system of S. mansoni have great potential as targets for antischistosomal drugs aimed at disrupting parasite mobility. In addition, targeting GPCRs that were shown to be expressed in the gonads of male or female schistosomes may result in disruption of the reproduction process. This is desirable because schistosome eggs are the main cause for pathology in schistosomiasis. Furthermore, this paper has shown that schistosome GPCRs are expressed in various life-stages. Multiple receptors were shown to be upregulated in the juvenile life-stage, indicating that drugs targeting these GPCRs would be effective against schistosomula. This paper has found that seven S. mansoni GPCRs have been functionally characterized. These include two histamine-responsive (SmGPR-1, SmGPR-2) and two dopamine-responsive (SmGPR-3, SmD2) receptors as well as one acetylcholine-responsive (SmGAR), one glutamate-responsive (SmGluR) and one serotonin-responsive receptor (Sm5HTR). A part of these deorphanized receptors signal through atypical pathways. The majority of the deorphanized receptors have different pharmacological profiles than those of receptors in the mammalian host. This indicates that schistosome GPCRs have great potential for parasite- selective drug targeting. The first library screen of a flatworm GPCR has been reported and

121 indicated that structure-activity profiling can provide a deeper understanding of pharmacophores that are selective for parasite receptors.240

Schistosomiasis is one of the most devastating tropical diseases, affecting at least 230 million people.63 Currently, no vaccine exists to treat this disease and the only drug available is praziquantel (PZQ). This drug is inactive against juvenile schistosomes and the first signs of resistance of S. mansoni to PZQ have been observed.8,9,10 GPCRs are promising antischistosomal drug targets because schistosome GPCRs are expressed in various life-stages, both sexes and various tissues, including the gonads. Therefore, compounds that target these receptors can be used to combat the acute as well as the chronic phase of the parasitic disease, and prevent the dissemination of schistosome eggs.

Future studies will need to be undertaken to further exploit GPCRs for the anthelmintic drug discovery. The majority of the described GPCRome remain to be functionally characterized. These can be characterized using the aforementioned assays, including cell-based signaling assays that measure changes in Ca2+ or cAMP following receptor activation. In addition, yeast assays have been used successfully to characterize schistosome GPCRs. Future studies could also use the recently reported nanoluciferase-based complementation assay.227 Another novel method that has been described is the GloSensor assay, which allows the profiling of schistosome GPCRs in a high throughput screening format.168 Future investigates could also modulate this assay so that the activity of receptors other than Gi and Gs-coupled GPCRs can be monitored. Furthermore, the recently described Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR- Y2H) may be useful to detect S. mansoni GPCR–ligand interactions and deorphanize S. mansoni receptors.85

The availability of immortalized cell lines for schistosomes would greatly benefit future studies. This may be established by using isolated cells with stem cell characters.65,241 This would be especially useful because heterologous expression of schistosome GPCRs often remains challenging. Integration of molecular approaches with whole animal studies will result in the identification and validation of promising antischistosomal drug targets.28 RNAi studies will continue to develop and will provide important information about the biology of GPCRs in S.

122 mansoni. Compound libraries can be screened against heterologously expressed schistosome receptors and structure-activity profiling will provide a deeper understanding of pharmacophores that are selective for specific parasite receptors.239 Subsequently, hits that emerge from in vitro screening can be tested in vivo and chemically-optimized to improve its selectivity towards schistosomes.

A deeper understanding of the biology of S. mansoni, and a target-based screening method integrated with whole worm screening approaches will advance antischistosomal drug discovery. Moreover, exploitation of the great evolutionary difference between GPCRs in S. mansoni and those of the mammalian host will aid in the development of parasite-selective, antischistosomal drugs.

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