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 by 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. 2 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 3 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. 4 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. 5 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