G PROTEIN-COUPLED RECEPTOR DE-ORPHANIZATION TO IDENTIFY NOVAL FLATWORM DRUG TARGETS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0213129
• Project Start Date: Nov 1, 2007
• Project End Date: Oct 31, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011

Performing Department
VETERINARY MEDICINE

Non Technical Summary
Parasitic flatworms, both flukes and tapeworms, continue to pose a significant challenge to both animal and human health. The clinician's pharmacological toolbox for treating flatworm infestations is very meager, both for human and veterinary disease. Against that backdrop, reports of resistance to praziquantel, the drug of choice for treating many trematode and cestode infections, are alarming. New drugs against flatworm infections are needed. G protein-coupled receptors (GPCRs) are cell surface proteins which are long-established, lucrative targets for therapeutic intervention; drugs targeting GPCRs account for 50-60% of all prescription pharmaceuticals. Recently, there has been very rapid progress on resolving the genomes of a number of important flatworm--including the most important parasitic flatworms of man (Schistosoma mansoni) and animals (Fasciola hepatica), as well as the most important model, free-living flatworm (Schmidtea mediterranea). The rapidly expanding flatworm genomic resources provide a timely opportunity to identify a large number of flatworm GPCRs, many of which will prove to be somewhat unique to invertebrates and, therefore, attractive targets for new drugs targeting flatworms. A limiting factor in determining the potential of exploiting newly discovered GPCRs is the determination of the endogenous transmitters with which they interact, or pairing native ligands with these GPCRs. This process is called "de-orphanization" of those receptors. The central goal of this project is to use a novel approach to de-orphanize a large number of flatworm G protein-coupled receptors (GPCRs), and to identify a subset of these as GPCRs responsive to neuropeptide ligands. Our hypothesis is that the ligands for some of these GPCRs are flatworm neuropeptides. The completion of this project will validate a novel approach to pairing GPCRs with their native transmitters, an approach which is markedly more efficient and amenable to larger-scale projects. The completion of this project will also result in the identification of some flatworm neuropeptide GPCRs. The identification of flatworm neuropeptide GPCRs is tantamount to the identification of attractive potential anthelmintic drug targets, since flatworms and other early-diverging invertebrates have unique families of neuropeptides which are not present in mammals.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
313	3310	1110	90%
315	3310	1180	10%

Knowledge Area
313 - Internal Parasites in Animals; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1180 - Pharmacology; 1110 - Parasitology;

Keywords

flatworms, parasites, helminths, g protein-coupled receptors,

receptors, neuropeptide, schistosome, fluke, fasciola, chemotherapy

Goals / Objectives
Parasitic flatworms, both flukes and tapeworms, continue to pose a significant challenge to both animal and human health. The clinician's pharmacological toolbox for treating flatworm infestations is very meager, both for human and veterinary disease. Against that backdrop, reports of resistance to praziquantel, the drug of choice for treating many trematode and cestode infections, are alarming. New drugs against flatworm infections are needed. G protein-coupled receptors (GPCRs) are cell surface proteins which are long-established, lucrative targets for therapeutic intervention; drugs targeting GPCRs account for 50-60% of all prescription pharmaceuticals. The rapidly expanding flatworm genomic resources provide a timely opportunity to identify a large number of flatworm GPCRs, many of which will prove to be somewhat unique to invertebrates and, therefore, attractive targets for new drugs targeting flatworms. The central goal of this project is to use a novel approach to identify the cognate ligands for a large number of flatworm G protein-coupled receptors (GPCRs), and to identify a subset of these as GPCRs responsive to neuropeptide ligands. Our hypothesis is that the ligands for some of these GPCRs are flatworm neuropeptides. The completion of this project will validate a novel approach to pairing GPCRs with their cognate ligands, an approach which is markedly more efficient and amenable to larger-scale projects. The approach does not require full-length cloning, it does not require heterologous receptor expression, and it allows for the screening of the receptors in the presence of their endogenous complement of accessory and signaling proteins. The completion of this project will also result in the identification of some flatworm neuropeptide GPCRs. The identification of flatworm neuropeptide GPCRs is tantamount to the identification of attractive potential anthelmintic drug targets, since flatworms and other early-diverging invertebrates have unique families of neuropeptides which are not present in mammals. The central goal of this project is to use a novel approach to identify the cognate ligands for a large number of S. mediterranea GPCRs, and to identify a subset of these as GPCRs responsive to neuropeptide ligands. Our hypothesis is that the ligands for some of these GPCRs are flatworm neuropeptides. One goal of this project is the validation of an approach to GPCR de-orphanization which is more amenable to larger scales and is more efficient. * Our approach does not require cloning of the full-length GPCR. RNAi can work with dsRNA targeting only a fraction of a gene transcript. * Our approach does not require heterologous expression. * Our approach assays GPCR signaling with the receptor in its native cells, along with its natural complement of accessory and signaling proteins. The other primary goal is to identify and characterize flatworm neuropeptide receptors, which are attractive targets for much needed, novel anthelmintics.

Project Methods
Bacteria-mediated RNAi protocol This RNAi protocol is a derivative of a bacterial-feeding protocol in wide use for both C. elegans and S. mediterranea (Timmons et al. 2001; Reddien et al. 2005). Receptor de-orphanization protocol To determine downstream-signaling of silenced putative GPCRs and their ligands in S. mediterranea, whole worm-derived cell suspensions will be used in high throughput assays (96 well plates) to measure cellular cAMP and IP3 levels. The CatchPoint cAMP Fluorescent Assay (Molecular Devices) will be used to determine cAMP levels, while the IP-One HTRF Assay (Cisbio) will serve the same purpose for IP3. Both of these assays are applicable to a worm cell suspension which we routinely generate in the laboratory.

Progress 11/01/07 to 10/31/10

Outputs
OUTPUTS: This project resulted in the identification of over 400 G protein-coupled receptors in two different flatworms, the parasite Schistosoma mansoni and the free-living flatworm Schmidtea mediterranea. The schistosome receptors are made publicly-available in the Nature publication of the schistossome genome (Nature ...). The Schmidtea receptors are part of the manuscript we are submitting to Genome Biology this week. We have also developed reliable gene-silencing protocols using dsRNA delivered by E. coli ingestion for silencing some of these GPCRs in Schmidtea. We have used these to silence putative G protein-coupled Acetylcholine Receptors (GARs) and serotonin receptors. These protocols have been communicated to our scientific community via oral presentations at the International Congress of Parasitology (Melbourne, Australia) and Regulatory Peptides Meetings (Belfast, Northern Ireland) where we gave invited presentations. Most importantly, we have validated our proposed approach of using an RNAi-based approach to de-orphanize GPCRs in flatworms. Specifically, we have identified putative serotonin receptors and used RNAi to demonstrate that cyclic AMP responses observed in response to serotonin are dependent on those receptors. PARTICIPANTS: Tim A. Day, Professor, Dept of Biomedical Sciences, ISU Mostafa Zamanian, Graduate Student, ISU. Mostafa adopted an E. coli feeding protocol for silencing GPCRs in free-living flatworms. He has worked with Paul McVeigh of Queen's University Belfast to adopt it so schistosomes. Zamanian also tavelled to Jonathan Marchant's laboratory at University of Minnesota to learn specialized techniques for in situ hybridization of GPCRs in flatworms. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Other laboratories are utilizing the receptor sequences that we have published, and we have communicated our gene-silencing protocols to other laboratories as well. We know of at least two other laboratories that are using those protocols.

Publications

• M. Zamanian, M.J. Kimber, P. McVeigh, A.G. Maule and T.A. Day (submitted, 2011). The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea. Genome Biology.
• M. Berriman, B. Haas, P.T. LoVerde, R.A. Wilson, G.P. Dillon, G.C. Cerqueira, S.T. Mashiyama, B. Al-Lazikani, Luiza F. Andrade, P.D. Ashton, M.A. Aslett, D.C. Bartholomeu, G. Blandin, C.R. Caffrey, A. Coghlan, R. Coulson, T.A. Day, A. Delcher, R. De Marco, A. Djikeng, T. Eyre, J.A. Gamble, E. Ghedin, Y. Gu, C. Hertz-Fowler, H. Hirai, Y. Hirai, R. Houston, A. Ivens, D.A. Johnston, D. Lacerda, C.D. Macedo, P. McVeigh, Z. Ning, G. Oliveira, J.P. Overington, J. Parkhill, M. Pertea, R.J. Pierce, A.V. Protasio, M.A. Quail, M.A. Rajandrean, J. Rogers, M. Sajid, S.L. Salzberg, M. Stanke, A.R. Tivey, O. White, D.L. Williams, J. Wortman, W. Wu, M. Zamanian, A. Zerlotini, C.M. Fraser-Liggett, B.G. Barrell, N.M. El-Sayed (2010). The Genome of the blood fluke Schistosoma mansoni. Nature 460:352-358.
• P. McVeigh, G.R. Mair , L. Atkinson, M. Zamanian, P. Ladurner, E. Novozhilova, N.J. Marks, T.A. Day, A.G. Maule (2009). Discovery of multiple neuropeptide families in the phylum Platyhelminthes. International Journal for Parasitology 39:1243-1252.
• Paul McVeigh, Michael J Kimber, Ekaterina Novozhilova, Louise Atkinson, Alan P Robertson, Mostafa Zamanian, Nikki J Marks, Aaron G Maule and Tim A Day (2010). Neuropeptide signalling in schistosomes. International Congress of Paarasitology, Melbourne, Australia.
• G protein-coupled receptors in two platyhelminths: Schistosoma mansoni and Schmidtea mediterranea (2009). Zamanian M., Kimber M.J., McVeigh P., Maule A.G. and Day T.A. British Society for Parasitology, Edinburgh, UK.