Deorphanization and Characterization of Neuropeptide Receptors in the Model Nematode Caenorhabditis Elegans PDF Download

Author: Elizabeth Ruiz Lancheros
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Languages : en
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Book Description
"Nematode neuropeptides play critical roles in modulating neuronal networks acting independently or together with other neurotransmitters; consequently, they influence motor programs and all nematode behaviour outputs. The FMRF-amide like peptide (FLP) family of neuropeptides is particularly well-conserved across the phylum Nematoda and differs from its vertebrate counterpart. Peptides in this family are involved in neuromuscular functions, feeding/metabolism, and reproductive behaviors in free-living and parasitic nematodes. These unique characteristics of FLPs make their endogenous receptors, mainly G protein-coupled receptors (GPCRs), potential targets for the rational discovery of chemotherapeutic agents that target parasitic nematodes. Relatively few FLPs have been associated with a receptor and several important candidate neuropeptide-GPCRs remain orphans (unpaired with its endogenous ligand). In this work, we designed an in situ deorpanization strategy in the model free living nematode Caenorhabditis elegans. Based on the conservation of FLPs, it is expected that identification of receptors in C. elegans will enable the identification and characterization of homologous receptors in parasitic species. To validate the in situ strategy, we first developed an alternative bioassay to allow the characterization of neuroactive FLPs in C. elegans. A proof-of-concept study using the novel anthelmintic derquantel revealed that a cut-worm model can be used to remove the cuticle as a permeable barrier, allowing compounds like derquantel to reach their site of action and induce responses controlled by the neuromuscular system. Consistent with these observations, selected FLPs induced a range of locomotor effects (phenotypes) in cut-preparations. Based on this, FLP-phenotypes were used as a read-out method to screen candidate GPCRs in cut-preparations. Experiments with the ligand/receptor pair FLP18-6 (DVPGVLRFa)/NPR-5 revealed that an npr-5 knockout strain loses the phenotype induced by FLP-18 in wild type (wt) cut-preparations, and this was reversed by reintroducing NPR-5. These findings suggest that mutations in NPR-5 specifically disable the FLP-18 phenotype and validated the use of knockout strains with loss-of-function mutations in GPCR genes for in situ deorphanization using the cut-worm model. By performing a low-throughput screen of receptors for 7 FLPs in 28 knockout strains, 9 novel FLP-GPCR associations were identified; one of them was further characterized in a yeast expression system. Some FLPs were associated with more than one GPCR and one GPCR with more than one FLP in situ. This promiscuity becomes relevant in the rational search of drugs that target deorphanized FLP-GPCRs and impair parasite viability. To investigate the receptor interaction with endogenous ligand(s) and gain insight into how two receptors interact with the same ligand we used the cut-worm model and receptors expressed in yeast for structure-activity relationship studies. Results for the FLP-18 receptors NPR-4 and NPR-5 revealed that the FLP18-6 C-terminus is essential for receptor activation in vitro and peptide activity in the cut-worm model, more than N-terminus. The use of FLP18-6 alanine analogs confirmed these observations, identified the key role of specific peptide residues for receptor activation, and suggested a differential interaction of peptide side chains with each receptor. This information is valuable for further development of promiscuous non-peptide ligands that act on both receptors. Overall, the results presented here provide invaluable data on FLP-GPCR associations and interactions, as well as in situ and in vitro bioassays useful for ongoing efforts in anthelmintic discovery and neuropeptide GPCR characterization." --