Characterization of serotoninergic G-protein coupled receptors from cestodes: new potential targets for drugs against neglected tropical diseases

CAMICIA, F.; CHAN, JOHN D.; WOODHOUSE, KRISTEN; MARCHANT, JONATHAN S.; PARK, SANG KYU; ROSENZVIT, M.C.

Buenos Aires

Congreso; Drug Discovery for Neglected Diseases International Congress 2018 4th Scientific Meeting of ResNet NPND; 2018

Instituto de la Química y Metabolismo del Fármaco, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires

Abstract: Echinococcus canadensis is a platyhelminth parasite that belongs to the class Cestoda and is the etiological agent of the Hydatid disease, a neglected disease that affects public health and the economy in Argentina and worldwide. Currently, the treatment for echinococcosis and other cestode infections in humans relies on benzimidazoles, mainly albendazole, which is used alone or in combination with praziquantel. However, the scarcity of anthelmintic drugs available and the emergence of resistant parasites, makes the discovery of new anthelmintic drugs an imperative need. To tackle this problem, we propose to characterize G-protein coupled receptors from cestodes as new pharmacological targets. In our previous work1, we found that serotoninergic GPCRs (5-HT GPCRs) are of major importance in cestode movement and showed distinctive pharmacological features. Objective: the aim of this work was to study the function of a new 5-HT GPCR from Echinococcus canadensis. The ortholog of this gene was also found in another cestode called Mesocestoides corti and this suggests that this gene is conserved in other members of the class and could perform important roles beyond Echinococcus spp species. Material and methods: Bioinformatics analyses suggest the existence of genes encoding for 5-HT GPCRs and this information was used for the design of primers. New cDNA was synthesized from reverse transcription using RNA extracted from protoscoleces of pig origin as a template for PCR reactions. The amplificated cDNA coding for the serotoninergic gene was cloned, sequenced and finally used for transient transfections in a stable cell line of HEK293 cells expressing GNA15. GNA15 encodes for a promiscuous G protein which increases intracellular calcium levels when it couples with a functional receptor. Calcium levels were measured using a Fluorescent Imaging Plate Reader (FLIPR) assay2. Results: When the cell line HEK293-GNA15 was transfected with a gene encoding for the cestode receptor, the calcium levels increased only in the presence of serotonin but not in the presence of other ligands like tryptamine, tyramine, octopamine, acetylcholine, histidine or dopamine. Discussion: The dataset confirm the bioinformatics analyses showing that the cloned gene encodes for a new 5-HT GPCR conserved in cestodes. Conclusions: The cloning strategy followed by sequencing and expression in HEK cells revealed a new 5-HT GPCR. The results obtained confirm bioinformatics predictions and will be tested as a target for cestocidal drugs. References1.Camicia F, Celentano AM, Johns ME, Chan JD, Maldonado L, Vaca H, Di Siervi N, Kamenetzky L, Gamo AM, Ortega-Gutierrez S, Martin-Fontecha M, Davio C, Marchant JS, Rosenzvit MC. Unique pharmacological properties of serotoninergic G-protein coupled receptors from cestodes. PLoS Negl Trop Dis. 2018 Feb 9;12(2):e0006267.2.Harvey JH, van Rijn RM, Whistler JL. A FLIPR assay for evaluating agonists and antagonists of GPCR heterodimers. Methods Mol Biol. 2013;995:43-54.