Deorphanization and characterization of neuropeptide receptors in
the model nematode, Caenorhabditis elegans
Elizabeth Ruiz Lancheros
Institute of Parasitology
McGill University
Montreal - Canada
April 2014
A thesis submitted to Graduate and Postdoctoral Studies office in partial fulfillment of
the requirements for the degree of Doctor of Philosophy
© Elizabeth Ruiz Lancheros 2014
Abstract
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 cutpreparations. 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
ii 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.
iii Résumé
Chez les nématodes, les neuropeptides jouent un rôle essentiel dans la modulation des réseaux
neuronaux. En agissant indépendamment ou conjointement avec d’autres neurotransmetteurs, ils
influencent les programmes moteurs ainsi que toutes les réponses comportementales chez ces
organismes. Les FMRF amide-like peptides (FLPs), font partie d’une famille de neuropeptides
particulièrement conservés dans l’embranchement phylogénétique des nématodes et qui
divergent de leurs équivalents chez les vertébrés. Les FLPs sont impliqués dans le contrôle des
fonctions neuromusculaires, la régulation de l’alimentation et du métabolisme, les
comportements liés à la reproduction chez les nématodes vivant seules où parasitaires. Ces
caractéristiques des FLPs font en sorte que leur récepteurs endogènes, principalement des
récepteurs couplés aux protéines G (G protein-coupled receptors; GPCR), pourraient constituer
d’excellentes cibles pour la conception d’agents chimiothérapiques contre les nématodes
parasitaires. Cependant, très peu de FLPs ont été appariés à un récepteur neuropeptidique et par
conséquent, plusieurs des GPCRs qui seraient des cibles potentielles sont orphelins, c’est-à-dire
non-appariés à leur ligand endogène. Dans cette thèse, nous avons développé une stratégie afin
d’apparier
certains
GPCRs
orphelins
à
leur
ligand
endogène
dans
le
nématode
Caenorhabditis elegans. Pour caractériser les FLPs neuroactifs chez C. elegans, nous avons mis
au point un test de dosage biologique en utilisant le derquantel, un nouvel anthelminthique, qui
fut appliqué sur des vers incisés. Les résultats obtenus avec cette approche montrent que ce
composé surmonte la barrière imperméable de la cuticule, atteint son site d’action et induit des
réponses sous le contrôle du system neuromusculaire. Lorsque différents FLPs sont testés selon
la même approche, nous observons alors qu’un certain nombre d’entre eux provoquent une
variété d’effets locomoteurs. Les phénotypes locomoteurs spécifiques aux différents FLPs furent
utilisés pour effectuer un criblage des différents GPCRs candidats. En exploitant le même
modèle expérimental avec le couple ligand/récepteur FLP18-6 (DVPGVLRFa)/NPR-5 déjà
identifié, nous observons que la souche dont le gène npr-5 est inactivé par knockout, perd de
manière spécifique le phénotype induit par FLP-18 comparativement à la souche sauvage. En
effet, ce phénotype est rétabli lorsqu’une copie intacte du gène NPR-5 est réintroduite dans la
iv souche inactivée. Ces résultats prouvent que l’utilisation de souches comportant une inactivation
de gènes encodant pour des GPCRs peut servir à apparier in situ, dans les préparations de vers
incisés, des récepteurs orphelins à leurs ligands endogènes. Grâce à un criblage à faible débit de
7 FLPs contre 28 souches inactivées pour de récepteurs GPCRs, 9 nouvelles associations FLPGPCR furent identifiés; l’une de ces paires fut caractérisée avec plus de détail in vitro dans un
système d’expression chez la levure. Certains de ces FLPs ont été associés avec plus d’un GPCR,
tandis qu’un GPCR a été associé avec plus d’un FLP. L’existence de ces multiples associations
entre ligands et récepteurs devra donc être prise en considération lors de la recherche de
composés ciblant les couples FLP-GPCR qui compromettent la viabilité d’un parasite. Pour
mieux comprendre comment un récepteur interagit avec son ligand endogène, et comment deux
récepteurs peuvent interagir avec un même ligand, nous avons étudié leur relation structureactivité dans les modèles de vers incisés et d’expression de récepteurs dans la levure. Avec ces
deux approches, nous observons que la portion C-terminale plutôt que la portion N-terminal de
FLP18-6 est essentielle pour l’activation des récepteurs NPR-4 et NPR-5. L’utilisation
d’analogues peptidiques de FLP18-6 pour lesquels les acides aminés ont été séquentiellement
remplacés par la L-alanine confirme ces observations. De plus, une interaction différentielle fut
identifiée entre les chaînes latérales d’acides aminés et chacun de ces récepteurs. En somme, les
résultats présentés dans cette thèse fournissent de précieuses informations au sujet des différents
types d’interactions entre FLP-GPCR. Ils montrent également que les approches in situ et in vitro
que nous avons développées, soit les modèles de vers incisés et le système d’expression chez la
levure, sont tous deux viables pour la caractérisation de GPCRs neuropeptidiques. Notre
recherche apporte une nouvelle contribution aux efforts en cours pour le développement de
nouveaux d’agents vermifuges.
v Acknowledgments
There is no doubt that pursuing my doctoral studies has been a great challenge in several aspects
of my life. I am really grateful to life for giving me the opportunity to learn during these years,
not only about parasites, but more about myself, and for making me a stronger and more
persistent person to overcome any difficulty. Certainly, I could have not succeeded in my studies
without the help and support of several people that I would like to acknowledge.
I’m very grateful to my supervisor Dr. Timothy G. Geary for his invaluable academic and
personal support. I really thank him for being more than an academic mentor, for believe in my
intellectual capacities and for being a great teacher. I feel blessed and honored for having the
opportunity to be Tim’s student and to learn from him, not only good science but to be a better
human being as he is. I will always remember and appreciate his kindness, his advice and his
ability to see the positive in everything and to care about everyone.
I would like to extend my appreciation to the former and current members of the Geary-Beech
Lab for such a wonderful and friendly working and learning environment, you make going to the
lab a fun and enjoyable experience. Special thanks to my friend Dr. Yovany Moreno, who
suggested me to join Geary lab and was always there to help me and give me great advice, and to
Dr. Vanessa Dufour for all these years working together.
I also would like to show my deep gratitude to those who helped me in my research project:
Thomas Duguet for his advice and great help with C. elegans microinjection, Serge Dernovici
and Kathy Keller for their continuous technical support and kindness, Vicky Muise and Damian
Clarke for their help with yeast cultures, and Charles Viau and Dr. Tita Walter for assisting me
in the intense C. elegans dissections. Many thanks to my former students, Abdel Francis, Amy
Smith-Dijak and Maude Dagenais for giving me the opportunity to learn how to teach what I had
learned all these years. Thanks to Dr. Imad Baakini and for helping me with the translation of the
abstract to French.
vi Special thanks go to other members of the Institute of Parasitology, Drs. Paula Ribeiro and
Robin Beach for their continuous guidance, encouragement and excellent recommendations on
my research. Special thanks to Drs. Marie-Claire Rioux, Normand Cyr, Vijayaraghava Rao,
Fouad El-Shehabi, and Catherine Bourguinat for their great advice and discussions, and for being
a source of admiration and motivation. Sincere thanks also to Shirley Mongeau, Christiane
Trudeau and Michael Massé for their excellent assistance and kindness.
I’m also really grateful to other people at McGill, Dr. Joseph Dent for his valuable contributions
to my project and the members of his lab for advices in C. elegans work. Thanks to Dr. Gregor
Jansen for his guidance in yeast studies, and Drs. Terry Hebert and Dominic Devost for their
friendly help and support with cell cultures. Many thanks to the McGill OSD team for their great
academic support, especially to Miss. Patricia Diaz del Castillo for her kind advice.
Finally, I want to thank my friends and relatives in Canada and Colombia for their unconditional
support. Merci à Imad et Aurélie pour être ma famille à Montréal et pour prendre soin de moi.
Infinitas gracias a mis amigos colombianos en Canadá, especialmente a Yolima, Juan y Edna por
cuidar siempre de mi y por su invaluable amistad. Gracias totales a mis amigos y familiares en
Colombia quienes me han acompañado desde la distancia en esta gran aventura en las tierras del
norte. Finalmente, dedico este trabajo a mis padres quienes han trabajado incansablemente toda
su vida por su familia y por ayudarnos a cumplir nuestros sueños. Ellos han sido el mejor
ejemplo de constancia y dedicación que he tenido, gracias a su incondicional apoyo he
culminado todas mi metas.
vii Statement of Originality
The following aspects described in this thesis are considered contributions of original
knowledge:
Manuscript I. Ruiz-Lancheros, E., Viau, C., Walter, T. N., Francis, A., Geary, T. G, 2011.
Activity of novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans. Int. J.
Parasitol. 41, 455-461.
In this manuscript, we investigated whether the cuticle is a barrier for the activity of the novel
nicotinic acetylcholine receptor (nAChR) ligand derquantel (2-desoxoparaherquamide, DOPH),
in C. elegans. A direct and marked effect of derquantel on motility was observed using a cutworm model and its activity as antagonist of the action of different nAChR agonists was
documented in this model. This report provides the first evidence for the presence of the
derquantel molecular target in C. elegans and it is the first time that its possible interaction with
the novel nAChR agonist monepantel is assessed. Results suggest that derquantel lacks affinity
for the monepantel-sensitive nAChR subtype and that the drugs do not engage in a negative
interaction in C. elegans. This study also served as a proof-of-concept study to investigate
whether the barrier properties of the C. elegans cuticle can be overcome in a low-throughput
bioassay to characterize neuroactive peptides in situ.
Manuscript II. Ruiz-Lancheros, E., Viau, C., Walter, T. N., Dent, J. A., Jansen, G., Thomas, D.
Y., Geary, T. G. Novel approach for receptor deorphanization in the model nematode,
Caenorhabditis elegans. In preparation.
In this manuscript, we designed and validated an in situ strategy to pair physiologically relevant
FMRFamide-like peptides (FLPs) with endogenous C. elegans G protein-coupled receptors
(GPCRs). Using a cut-worm model, we described for the first time FLP locomotor effects on C.
elegans, which were used to screen for candidate GPCRs directly in C. elegans bioassays.
Novelty was generated by the use of FLP effects as baits to identify endogenous receptors in situ
viii instead of using heterologously expressed receptors. This deorphanization (matching) approach
allowed identification of 9 new FLP-GPCR associations that were undetected by reverse
pharmacology in previous studies. This is the first time that endogenous receptors for profoundly
active Ascaris suum peptides are proposed and that activation of a GPCR for the potent and
broadly distributed peptide KHEYLRFa (AF2) is documented. The new associations presented in
this report constitute important data for characterization of parasitic nematode FLP-GPCRs and
enhance the search for non-peptide ligands for these receptors as anthelmintic candidates.
Manuscript III. Ruiz-Lancheros, E., Geary, T. G. Structure-activity relationships of
Caenorhabditis elegans FLP-18 on the G protein-coupled receptors NPR-4 and NPR-5. In
preparation.
In this manuscript, we investigate the pharmacological bases of receptor interaction with an
endogenous ligand by structure-activity relationship (SAR) studies. This report constitutes the
first time that the interaction of DVPGVLRFa (FLP18-6) with its receptors NPR-4 and NPR-5
is assessed by SAR studies in C. elegans bioassay and in vitro using the heterologously
expressed receptors in Saccharomyces cerevisiae. Results from this study gave new insights into
how the FLP-18 C-terminus interacts with both receptors and the key roles of P3 and V5 for
receptor activation. Results suggest that NPR-4 and NPR-5 recognize FLP18-6 in a different
manner, which can account for differences in the receptors binding site and explain the
promiscuity of FLP-18 peptides.
Appendix, Manuscript IV. Larsen, M. J., Ruiz-Lancheros, E., Williams, T., Lowery, D. E.,
Geary, T. G., Kubiak, T. M., 2013. Functional expression and characterization of the C. elegans
G-protein-coupled FLP-2 Receptor (T19F4.1) in mammalian cells and yeast. Int. J. Parasitol.
Drugs Drug Resist. 3:1-7, doi: 10.1016/j.ijpddr.2012.10.002.
In addition to the work presented in the body of this thesis, we confirmed the association of FLP2 peptides (-EPLRFa) with the GPCR encoded in the gene T19F4.1 by its expression in
ix heterologous systems. We improved the pharmacological study of this receptor in mammalian
cells previously reported by Mertens, I., et al, (Biochem. Biophy. Res. Commun. (2005), 330,
967-974). We also expressed and characterized the FLP-2 receptor in a yeast recombinant
system, which allowed its inclusion in a high-throughput screen for small molecule ligands as
potential anthelmintics. Result suggested that FLP-2/T19F4.1 is a physiologically relevant
ligand/receptor pair and that receptor signalling through the Gαq pathway is conserved in
different heterologous systems.
x Contribution of Authors
The design and execution of experiments presented in this thesis were carried out by the author
with the supervision of Prof. Timothy G. Geary. In the first manuscript Abdel Francis
contributed by performing worm experiments under the supervision of the author. In the first and
second manuscripts, Charles Viau and Dr. Tita N. Walter performed cut-worm experiments also
under the supervision of the author. In the second manuscript, Dr. Joseph A. Dent contributed to
the design of rescue experiments and the analysis of data. Drs. Gregor Jansen and David Y.
Thomas designed the HOG assay and Dr. Gregor Jansen gave advice for its use in the
characterization of nematode GPCR expression in yeast. In the manuscript presented in the
Appendix, the author contributed by performing the pharmacological characterization of the
FLP-2 receptor in yeast and with the manuscript edition. This thesis was written by the author with
editorial contributions from Dr. Timothy G. Geary.
xi Table of Contents
Abstract.......................................................................................................................................... ii Résumé .......................................................................................................................................... iv Acknowledgments ........................................................................................................................ vi Statement of Originality ............................................................................................................ viii Contribution of Authors .............................................................................................................. xi Table of Contents ........................................................................................................................ xii Table of Figures.......................................................................................................................... xvi List of Tables ............................................................................................................................ xviii List of Abbreviations ................................................................................................................. xix Chapter I. Introduction and Literature Review ........................................................................ 1 1. Introduction .......................................................................................................................................... 1 2. Literature review .................................................................................................................................. 4 2.1. Caenorhabditis elegans neuroanatomy ....................................................................................... 4 2.2. Anthelmintic targets in the nematode nervous system ................................................................ 6 2.3. The neuropeptidergic signalling system ...................................................................................... 8 2.4. Neuropeptides ............................................................................................................................ 10 2.4.1. FMRF-amide like peptides (FLPs) distribution ........................................................................ 11 2.4.2. FLP general functions ............................................................................................................... 12 2.5. Neuropeptide receptors .............................................................................................................. 16 2.5.1. Neuropeptide GPCR identification ............................................................................................ 18 2.5.2. FLP-GPCR deorphanization ..................................................................................................... 19 2.6. FLP-GPCRs as drug targets ....................................................................................................... 23 References .................................................................................................................................................. 25 Connecting Statement I .............................................................................................................. 30 Chapter II. Manuscript I............................................................................................................ 31 Abstract...................................................................................................................................................... 32 xii 1. Introduction ........................................................................................................................................ 33 2. Material and methods ......................................................................................................................... 34 3. 4. 2.1. Cultures of C. elegans ............................................................................................................... 34 2.2. Drug solutions ........................................................................................................................... 35 2.3. Worm assays .............................................................................................................................. 35 Results ................................................................................................................................................ 36 3.1. Agonist concentration-response studies .................................................................................... 36 3.2. Cut worm assay with mecamylamine as an nAChR antagonist ................................................ 38 3.3. Effects of 2-DOPH on C. elegans ............................................................................................. 39 3.4. 2-DOPH is an nAChR antagonist .............................................................................................. 40 3.5. Interaction of 2-DOPH and AAD 1470 in cut worm assays...................................................... 41 Discussion .......................................................................................................................................... 42 Acknowledgments ..................................................................................................................................... 46 References .................................................................................................................................................. 46 Connecting Statement II............................................................................................................. 49 Chapter III. Manuscript II......................................................................................................... 50 Abstract...................................................................................................................................................... 51 1. Introduction ........................................................................................................................................ 52 2. Materials and Methods ....................................................................................................................... 53 3. 2.1. Materials .................................................................................................................................... 53 2.2. Worm assay and FLP phenotype identification ......................................................................... 54 2.3. Receptor selection ..................................................................................................................... 54 2.4. Receptor screen in C. elegans.................................................................................................... 55 2.5. Mutant rescue assay ................................................................................................................... 55 2.6. Receptor activation assay in heterologous system..................................................................... 55 2.7. Receptor expression and membrane localization in S. cerevisiae ............................................. 56 Results ................................................................................................................................................ 57 3.1. FLP-phenotype identification .................................................................................................... 57 3.2. Proof-of-concept ........................................................................................................................ 59 3.3. In situ screen .............................................................................................................................. 60 xiii 4. 3.4. Receptor activation in heterologous system .............................................................................. 63 3.5. Monitoring C. elegans GPCR expression and plasma membrane localization in yeast. ........... 65 Discussion .......................................................................................................................................... 66 Acknowledgments ..................................................................................................................................... 72 References .................................................................................................................................................. 72 Supplementary information ..................................................................................................................... 77 Connecting Statement III ........................................................................................................... 82 Chapter IV. Manuscript III. ...................................................................................................... 83 Abstract...................................................................................................................................................... 84 1. Introduction ........................................................................................................................................ 85 2. Materials and Methods ....................................................................................................................... 87 3. 2.1. Materials .................................................................................................................................... 87 2.2. In vitro heterologous expression in S. cerevisiae ...................................................................... 88 2.3. In situ C. elegans bioassay ........................................................................................................ 88 Results ................................................................................................................................................ 89 3.1. NPR-4 and NPR-5a heterologous expression in S. cerevisiae................................................... 89 3.2. FLP-18 analog effects in the in vitro bioassay .......................................................................... 90 3.2.1. Truncated analogs ..................................................................................................................... 90 3.2.2. Alanine substitutions ................................................................................................................. 91 3.3. 4. FLP18-6 and analog effects in C. elegans cut preparations ...................................................... 93 Discussion .......................................................................................................................................... 95 Acknowledgments ................................................................................................................................... 100 References ................................................................................................................................................ 100 Chapter V. General Discussion................................................................................................ 103 References ................................................................................................................................................ 109 Appendix. Manuscript IV. ....................................................................................................... 112 Abstract.................................................................................................................................................... 113 1. Introduction ...................................................................................................................................... 114 2. Materials and methods ..................................................................................................................... 115 xiv 3. 4. 2.1. Materials .................................................................................................................................. 115 2.2. Cloning and plasmid preparation ............................................................................................. 115 2.3. Cell culture, cell transfection and intracellular Ca+2 mobilization assay ................................. 116 2.4. Expression in yeast .................................................................................................................. 116 2.5. Data analysis ............................................................................................................................ 118 Results .............................................................................................................................................. 118 3.1. Molecular cloning of T19F4.1/FLP-2R and phylogenetic analysis ......................................... 118 3.2. Receptor expression and identification of cognate ligands for FLP-2R in mammalian cells .. 118 3.3. FLP-2R functional expression in yeast .................................................................................... 120 Discussion ........................................................................................................................................ 121 Acknowledgments ................................................................................................................................... 125 References ................................................................................................................................................ 125 xv Table of Figures
Chapter I. Introduction and Literature Review
Figure 1. G protein signalling pathways.. .................................................................................................. 17 Chapter II. Manuscript I.
Figure 1. Effects of pyrantel on cut Caenorhabditis elegans.. ................................................................... 37 Figure 2. Comparative effects of pyrantel on cut and intact Caenorhabditis elegans. .............................. 38 Figure 3. Antagonism by mecamylamine.. ................................................................................................ 39 Figure 4. 2-Desoxoparaherquamide (2-DOPH) paralyzes cut specimens of Caenorhabditis elegans ...... 40 Figure 5. 2-Desoxoparaherquamide (2-DOPH) antagonizes the actions of nicotinic acetylcholine receptor
(nAChR) agonists on cut specimens of Caenorhabditis elegans.. ..................................................... 41 Figure 6. 2-Desoxoparaherquamide (2-DOPH) and amino-acetonitrile derivative (AAD) 1470 do not
interact in cut specimens of Caenorhabditis elegans.. ....................................................................... 42 Chapter III. Manuscript II.
Figure 1. FLP phenotypes in wt and mutant cut worms............................................................................. 60 Figure 2. AF2-receptor screening.. ............................................................................................................ 61 Figure 3. AF1-receptor screening. ............................................................................................................. 62 Figure 4. Receptor activation in yeast by AF2. .......................................................................................... 64 Figure 5. Monitoring plasma membrane localization of heterologous GPCRs in yeast. ........................... 66 Supplementary information
Figure S1. AF8 and FLP3-1 receptor screening......................................................................................... 79 Figure S2. AF22, FLP13-3 and AF21 receptor screening.......................................................................... 80 Chapter IV. Manuscript III.
Figure 1. Concentration-response curves and EC50 values of FLP18-6 tested on NPR-4 and NPR-5a
receptors expressed in yeast. .............................................................................................................. 89 Figure 2. Concentration-response curves of N-terminally and C-terminally truncated FLP18-6 analogs on
receptors expressed in yeast. .............................................................................................................. 90 xvi Figure 3. Concentration-response curves of FLP18-6 N-terminal alanine modifications on receptors
expressed in yeast. .............................................................................................................................. 91 Figure 4. Concentration-response curves of FLP18-6 C-terminal alanine modifications on receptors
expressed in yeast. .............................................................................................................................. 92 Figure 5. Effects of FLP18-6 and analogs in C. elegans bioassay. ............................................................ 94
Appendix. Manuscript IV.
Figure 1. Alignment of predicted protein sequences of T19F4.1 and obtained clones. ........................... 119 Figure 2. Functional expression of FLP-2R/T19F4.1 in CHO cells; Ca2+ mobilization assay by FLIPR.
.......................................................................................................................................................... 120 Figure 3. FLP-2R/T19F4.1 activation by FLP-2 peptides in yeast. ......................................................... 121 xvii List of Tables
Chapter I. Introduction and Literature Review
Table 1. FLP physiological effects in A. suum and C. elegans. ................................................................. 13 Table 2. Deorphanized FLP-GPCRs by high-throughput screening. ......................................................... 22 Chapter III. Manuscript II.
Table 1. FLP-phenotypes in C. elegans preparations. ................................................................................ 58 Table 2. in situ deorphanized C. elegans GPCRs....................................................................................... 63 Supplementary information
Table S1. Selected receptors for screening. . ............................................................................................ 78 Chapter IV. Manuscript III.
Table 1. Analysis of concentration-response curves of FLP18-6 analogs tested on NPR-4 and NPR-5a.. 93 xviii List of Abbreviations
2-DOPH: 2-desoxoparaherquamide
5-HT: 5-hydroxytryptamine (Serotonin)
AAD: Amino-acetonitrile derivatives
ACh: Acetylcholine
AC: Adenylate cyclase
APF: Artificial perienteric fluid
AP: Aminopeptidase
bwRT: Body wall response type
CHO: chinese hamster ovary
CKR: Cholecystokinin-like receptor
CPE: Carboxipeptidase E
DA: Deamidase
DAG: Diacylglycerol
Emax: Maximum response
EP: Endopeptidases
ER: Endoplasmic reticulum
FLIPR: Fluorescence imaging plate reader
FLP: FMRF-amide (Phe-Met-Arg-Phe-NH2)–like peptide
flp: FMRFamide-like peptide precursor gene
FLP-2R: FLP-2 receptor
FRPR: FMRFamide peptide receptor
GΑΒΑ: γ-aminobutyric acid
gf : Gain-of-function
GEF: Guanine nucleotide exchange factor
GI: Gastrointestinal
GIRK: G-protein-regulated inward-rectifier K+
GluCl: Glutamate-gated chloride-channel
GNRR: Gonadotropin-releasing hormone receptor
G protein: GTP binding protein
xix GPCR: G protein-coupled receptor
GTP: Guanosine triphosphate
HOG: High-osmolarity glycerol
ISH: In situ hybridisation
INS: insulin-like peptides
IP3: Inositol 1,4,5-triphosphate
KO: Knockout
LDCV: Large dense-cored secretory vesicles
lf : Loss-of-function
MAP: Mitogen-activated protein
MAPKKK: MAP kinase kinase kinase
MLs: Macrocyclic lactones
NaCh: Na+ Channel
nAChR: Nicotinic acetylcholine receptor
NGM: Nematode growth medium
NMJ: Neuromuscular junction
NMUR: Neuromedin U receptor
NLP: Neuropeptide-like protein
NPR: Neuropeptide receptor
NTR: Nematocin receptor
ovRT: Ovijector response type
PAL: Peptidyl-α-hydroxyglycine α-amidating lyase
PHM: peptidylglycine-α-hydroxylating monooxygenase
PIP2: phosphatidylinositol 4,5-bisphosphate
PKA: Protein kinase A
PKC: Protein kinase C
PLC: Phospholypase C
SAM: Sterile α Motif
SAR: Structure-activity relationship
SPC: Subtilisin-like preprotein convertase
TKR: Tachykinin-like neuropeptide receptor
wt: Wild type
xx Chapter I. Introduction and Literature Review
1.
Introduction
Most commercially available chemotherapeutic agents used for controlling parasitic nematodes
that infect humans, companion and livestock animals compromise motor functions. For example,
ivermectin, levamisole, piperazine, pyrantel, emodepside, monepantel, and derquantel target
neurotransmitter receptors or ion channels in the nematode neuromuscular system and lead to
paralysis affecting locomotion, reproduction and/or feeding, and therefore impairing parasites
ability to sustain infection in the host. These drugs successfully control many different species of
parasitic nematodes and have broad-spectrum action since their targets are conserved in the
phylum Nematoda and can be pharmacologically distinguished from related host
neurotransmitter receptors and ion channels. Unfortunately, routine use of available
anthelmintics, similarities in their mechanism of action and the genetic characteristics of
nematodes has generated suboptimal responses to almost all available anthelmintics in parasite
populations. Consequently, it is an urgent task to find new classes of drugs to control parasitic
nematode infections that still affect more than a billion people and produce severe losses in
livestock production (Martin and Robertson, 2010).
The nematode neuropeptidergic component of the nervous system is an attractive source of
targets for rational discovery of new anthelmintics. Nematodes neuropeptides influence every
recognized worm behavior and have key roles in worm biology (Mousley et al., 2010). Some
neuropeptides, as well as other elements of the neuropeptidergic system, are broadly distributed
in the phylum and are distinctly different from their vertebrate counterparts. Particular attention
has been given to a specific family of neuropeptides, the FMRFamide-related or -like peptides
(FaRPs or FLPs), and their receptors over the past 20 years (McVeigh et al., 2006a). FLPs
potently influence motor function, have inter-phylum activities and are rarely found in
vertebrates. However, no currently available drug targets FLP receptors or any other component
of the neuropeptidergic system, and more work is needed to enable the use of modern high1 throughput screening technology to find new compounds that target this system in invertebrates
(McVeigh et al., 2012).
Finding endogenous neuropeptide receptors and characterizing their ligand-receptor interactions
are crucial steps to establish the essentiality of a particular neuropeptide signalling pathway in
worm biology and to consider it as a target for chemotherapy. Unfortunately, little information
on neuropeptide receptors in parasitic nematodes is available; most of the data comes from the
free-living nematode Caenorhabditis elegans. However, marked conservation of neuropeptide
families in the phylum suggests that the same conservation of their receptors across the phylum
Nematoda can be expected. Therefore, neuropeptide receptor characterization in C. elegans can
enable the identification of novel anthelmintic targets in parasites. To date, the deorphanization
process (pairing neuropeptide ligands with their cognate receptors) has relied on the heterologous
expression of neuropeptide receptors, especially G protein-coupled receptors (GPCRs), to be
used as baits for pools of synthesized neuropeptides in high-throughput formats. Despite intense
efforts in many laboratories over the past 15 years, most neuropeptide receptors remain orphan
(unpaired with an endogenous ligand) and new approaches are needed (Husson et al., 2007;
McVeigh et al., 2012).
Considering the pertinence of neuropeptidergic systems for drug discovery, the main initiative of
this work was to design an in situ strategy to match C. elegans putative FLP receptors with their
cognate ligands. Novelty was generated by the use of FLP ligands as baits in situ instead of in
systems in which receptors are heterologously expressed in mammalian cells, Xenopus oocytes
or yeast. In order to use C. elegans bioassays to study in situ molecules with low accessibility
through the cuticle, such as FLPs, we first developed an assay using a cut-worm model to
characterize the effects of nicotinic acetylcholine receptor (nAChR) agonists and antagonists in
C. elegans. Results of this initial test constitute the Chapter II of this document and show that
C. elegans cut preparations preserve motor activity and remove the cuticle as a permeability
barrier. In bisected C. elegans worms, otherwise impermeant ligands can reach their site of
action. Consequently, we established a cut-worm assay as being suitable to characterize
neuropeptide effects in situ.
2 In chapter III of this document, we focused on the design of a low-throughput screen for
matching FLP-GPCRs in situ using cut-worm preparations. We identified locomotion
phenotypes of a small number of FLPs in cut worms, and used them as a read out in a screen of
candidate FLP-GPCRs in C. elegans strains with loss-of-function (lf) mutations in individual
GPCR genes. Proof-of-concept experiments evidenced the specificity and selectivity of the in
situ strategy, enabling us to propose candidate GPCRs for 7 different FLPs. This new strategy
allows us to match FLP-GPCR pairs that have been missed by previous strategies using
heterologous expression systems. In addition, our different approaches to study the
pharmacology of newly deorphanized receptors in heterologous systems (manuscripts II and
IV), document the limitations of these systems to reproduce in situ observations and question
their use in high throughput deorphanization campaigns.
In the Chapter IV of this document (manuscript III), we build on the experience we gained in
in situ bioassays and heterologous expression systems (in vitro bioassays) to further characterize
ligand-receptor interactions by structure-activity relationship (SAR) studies. As an example, we
used a set of targeted analogs of the peptide FLP18-6 (DVPGVLRFa) and its C. elegans
receptors (NPR-5 and NPR-4) expressed in the yeast Saccharomyces cerevisiae. We observed
small differences in the way the receptors recognize residues in the FLP18-6 peptide and
identified key amino acids that are important for the interaction with each receptor. These new
insights into FLP18-6 interaction with its receptors at the molecular level can illuminate in silico
search for non-peptide ligands that target FLP-18 receptors acting as agonist or antagonists.
The data and analysis reported in this PhD thesis serve to accelerate the FLP receptor
deorphanization process and provide a better understanding of FLP-GPCR interactions in situ
and in vitro, which are top priorities for receptor identification and functional characterization.
Importantly, the strategies generated in this project have been used in an anthelmintic discovery
program that targets neuropeptide receptors in parasitic nematodes.
3 2.
Literature review
2.1.
Caenorhabditis elegans neuroanatomy
The free-living soil nematode C. elegans has been widely used to study the molecular
pharmacology and targets of anthelmintics that act in the neuromuscular system of parasitic
nematode species. One reason for this is that C. elegans has extensive neurological similarities
with parasite species in the phylum Nematoda. Broadly speaking, the nematode nervous system
consists of neuronal cells, their nerve-nerve junctions, the muscles they innervate, and all
proteins and signalling molecules involved in neurotransmission (Geary et al., 1992). The
neuroanatomy of C. elegans (neuronal morphology, connectivity and motorneuron distribution)
is essentially identical to that of the parasitic nematode Ascaris suum, and they employ the same
major neurotransmitters despite their phylogenetic distance (Holden-Dye and Walker, 2007).
Additionally, C. elegans is the only animal for which the entire "wiring diagram" of the nervous
system has been described (White et al., 1986). The nervous system of hermaphrodites is
distinguished by its apparent simplicity and versatility; it is limited not only in cell number (302
neurons and 56 support cells), but also in the number of processes or branches (Hall et al., 2006).
Neuronal cell bodies are mainly clustered in the head and tail, and along the body in longitudinal
nerve tracts at the ventral, subventral, lateral, subdorsal, and dorsal positions. They communicate
through around 6400 chemical synapses, 900 electrical synapses (gap junctions) and 1500
neuromuscular junctions (NMJ) (Riddle et al., 1997; Altun and Hall, 2011).
C. elegans neurons have simple monopolar or bipolar structures with few or no branches, unlike
the extensive neuronal arborizations typically found in higher animals. Somatic neurons in the
body wall and their processes are localized between the hypodermis and the muscle and are
separated from the muscle by a basal lamina. In addition and in contrast to arthropods and
vertebrates, the muscle cells of C. elegans extend elongated processes or arms to synapse with
motor neurons; several arms can aggregate to receive inputs from single neuron and form
chemical and electrical synapses between them (Von Stetina et al., 2006). Despite these
structural differences, most of the genes expressed in the C. elegans nervous system have
homologs in vertebrates, including those that encode neurotransmitters, biosynthetic enzymes,
4 neurotransmitter receptors and proteins that function in neurotransmitter release pathways. A
high percentage of neuronally expressed genes are involved in sensory recognition, neuronal
excitability and cell-cell communication (Bargmann, 1998), which makes C. elegans an excellent
model for neurological studies.
Despite the simplicity of the C. elegans nervous system, it controls several complex tasks. Basic
behaviors like locomotion, foraging, pharyngeal pumping and defecation are regulated by
chemosensory neurons, mechanosensory neurons and motorneurons. The animal displays
specific sexual behaviors like egg laying in hermaphrodites and mating behavior in males and is
also able to distinguish and respond to different kinds of chemicals, odors, temperature shifts and
sources of food. Most of these behaviors show high plasticity, are determined by food
availability and can be modified by learning and memory (Giles et al., 2006).
This versatility is possible because the nematode nervous system has diverse types of neurons
that are intrinsically different. Additionally, the final output, changes in behaviours, depends on
the changes produced by a high number of intracellular signalling molecules which compensate
for the structural limitations of the nervous system (Bargmann, 1998; Hall et al., 2006). As
signalling molecules, C. elegans employs most of the classical neurotransmitters found in
vertebrates, including acetylcholine (ACh), γ-aminobutyric acid (GABA), glutamate, serotonin
(5-HT, 5-hydroxytryptamine) and other monoamines.
ACh is the only neurotransmitter that is essential for C. elegans viability. It is released by
excitatory neurons and acts as the primary excitatory neurotransmitter for the control of motor
functions. ACh also controls pharyngeal pumping and mediates mechanosensation. GABA is
released by inhibitory motorneurons and interneurons and causes relaxation of body wall muscle
by hyperpolarization of muscle cell membrane potential. Glutamate acts as inhibitory transmitter
on pharyngeal muscle and sensory transmitter in the amphidial neurons. 5-HT stimulates eggslaying and pharyngeal pumping; it inhibits locomotion, defecation and the male-specific ventral
tail curling behaviour involved in mating. Other biogenic amines, including dopamine,
octopamine and tyramine, display distinct and overlapping roles in sensory and feeding
5 behaviours, locomotion, defecation and egg-laying (Geary et al., 1992; Brownlee and
Fairweather, 1999; Brownlee et al., 2000).
In addition to classical neurotransmitter, C. elegans uses a high number of neuropeptides that can
act as direct neurotransmitters, neuromodulators or neurohormones. Several neuropeptides have
been associated with different worm behaviours: neuromuscular functions (locomotion and
reproduction), mechano- and chemosensation, reproductive behaviors, social feeding and
gustatory associative learning and memory. Indeed, peptide signaling molecules appear to be
involved in almost every recognized behavior not only in C. elegans, but also in other helminths
(Kubiak et al., 2003a; Chalasani et al., 2010; Beets et al., 2012; Frooninckx et al., 2012; Garrison
et al., 2012). This evidence suggests that the neuropeptidergic system plays critical roles in
nematode biology.
2.2.
Anthelmintic targets in the nematode nervous system
Since behaviors like movement, feeding and reproduction are critical for proper location and
maintenance of parasitic nematodes in the host, the neuromuscular system has long been
identified as a major site for chemotherapy. Unsurprisingly most anthelmintics used for
controlling parasitic nematodes that infect humans, companion and livestock animals target
neuromuscular signalling, compromising nerve-muscle function to impair normal parasite
biology and to remove them from the host.
For example, levamisole and pyrantel are nicotinic agonists that selectively affect nicotinic
acetylcholine receptor (nAChR) on nematode muscle or neurons to produce spastic paralysis and
inhibition of egg-laying. These and other nicotinic agonists act selectively in different nAChR
subtypes and are used against gastrointestinal (GI) nematodes parasites. Nicotinic antagonists
such as paraherquamide, desoxyparaherquamide and phenothiazine also inhibit muscle motility
and induce paralysis, but have been less used due to their toxic effects and lack of commercial
formulations. On the other hand, piperazine acts as a GABA agonist, gating GABA channels to
induce an inhibitory effect that leads to flaccid paralysis in nematodes of the GI tract (Robertson
and Martin, 2007).
6 The broadly used ivermectin and other macrocyclic lactones (MLs) also target a conserved
neurotransmitter receptor in invertebrates. MLs selectively activate glutamate-gated chloridechannels (GluCls) that are present in different tissues in nematodes and arthropods but are absent
from vertebrates. Depending on the channel distribution in a particular parasite and the
sensitivity to MLs, MLs can inhibit pharyngeal pumping and feeding, leading to starvation, or
inhibit muscle motility, inducing long-lasting paralysis, or they can inhibit egg-laying and
consequently reproduction. Avermectins are quite potent, have a very broad-spectrum of action,
and are effective against GI and systemic (filariae) nematodes (Martin and Robertson, 2010).
Recent evidence suggests that ivermectin clears Brugia malayi microfilariae from the host by
acting on a GluCl present in the secretory pore, which disrupts processes that modulate the host
immune system by secreted proteins (Moreno et al., 2010).
The semi-synthetic cyclic depsipeptide emodepside is effective against parasites that are resistant
to other anthelmintics and in C. elegans produce impaired locomotion and inhibition of the
pharyngeal pumping and egg-laying. Studies in C. elegans, A. suum and H. contortus suggest
that the emodepside molecular targets include latrophilin-like receptors, but most prominently a
calcium-activated potassium channel (SLO-1) that is widely expressed in the C. elegans nervous
system and muscle at which emodepside probably exerts its primary inhibitory actions (HoldenDye and Walker, 2007). In contrast, the benzimidazoles, such as thiabendazole, albendazole,
triclabendazole and flubendazole, inhibit egg production and nematode survival. Benzimidazoles
act by binding to β-tubulin and inhibiting microtubule assembly, which affects intracellular and
vesicular transport, cell division and synaptic function (although paralysis is not a prominent
effect of these drugs).
Because ligand-recognition features of receptors and microtubule formation are highly conserved
in nematodes but have chemically exploitable differences between nematodes and mammals,
these empirically discovered drugs have been successful in selectively controlling multiple
species of parasitic helminths (and in some cases arthropods) despite their phylogenetic distance.
However, their intensive use has commonly generated parasite populations that are resistant to
them, defined as heritable suboptimal responses to standard doses (Kaplan, 2004). Several
aspects of pharmacology and therapeutics influence the development of anthelmintic resistance,
7 including the high mutation rate in nematodes, the high degree of polymorphism in their
genomes, the selection of resistant population by the routine and intensive use of the same
anthelmintics, as well as the selection of alleles with mutations in the drug target, in related
genes and/or in genes that improve fitness in presence of the drug. Differences in intrinsic
sensitivity of different drug target subtypes and the use of anthelmintics with the same targets or
overlapping mechanisms of action also foster the induction of resistance and cross-resistance
(Martin and Robertson, 2010).
Furthermore, currently available anthelmintics are suboptimal for use in some target species,
especially humans (Osei-Atweneboana et al., 2007; Diawara et al., 2009; Geary et al., 2010).
Factors underlying this situation include inadequate spectrum of action and insufficient efficacy
in dosing regimens compatible with mass drug administration campaigns.
Considering this perspective, the discovery of new anthelmintic drugs with novel mechanisms of
action (targets), and which escape current mechanisms of resistance and can reinvigorate the
treatment of the parasitic nematode infections, is an urgent need. Since the neuromuscular
system has provided most of the targets for the current broad-spectrum anthelmintics, the
druggability of the nematode neuromuscular system is unquestionable and can provide more
opportunities for target-based drug discovery (McVeigh et al., 2012). In addition, C. elegans is
sensitive to most anthelmintic drugs and their molecular targets and mechanisms of action have
been broadly studied in this species. Therefore, it is highly worthwhile to use C. elegans as a
model ‘parasite’ to study new molecular drug targets, taking advantage of the enormous variety
of molecular genetics techniques developed for studies in this organism (Holden-Dye and
Walker, 2007). As mentioned above, the neuropeptidergic component of the nervous system also
plays critical roles in the worm biology and is recognized to be a good repository of new drug
targets, although it remains to be exploited for chemotherapy (Maule et al., 2002).
2.3.
The neuropeptidergic signalling system
The neuropeptidergic signaling component of invertebrate nervous systems is highly developed
and complex. The system involves many different neuropeptides families, the neuropeptide
processing enzymes, the endogenous neuropeptide receptors and the molecules involved in
8 signaling cascades. Some components are present in vertebrates while others are exclusive to
invertebrates or even species-specific. Neuropeptide maturation occurs in a different fashion in
helminths compared to vertebrates; a single gene can encode multiples copies of the same or
different neuropeptides and the precursor molecule undergoes extensive posttranslational
processing in the trans-Golgi network prior to the production of individual neuropeptides.
Neuropeptide processing in nematodes involves digestion of a precursor protein by a subtilisinlike preprotein convertase (SPC) at specific dibasic motifs, digestion of the carboxyterminal
amino acid of the peptide-processing intermediates by a carboxypeptidase E (CPE), and
amidation of the glycine C-termini in two sequential steps by peptidylglycine-α-hydroxylating
monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL). This last
process protects neuropeptides from degradation and activates their transport in large densecored secretory vesicles (LDCV) to the nerve terminal where they are released by increasing
Ca2+ concentration throughout the nerve terminal. After a mature neuropeptide has been released
at the synapse and has performed its receptor-activating function, the signal is terminated by
enzymatic digestion by different aminopeptidases (AP), deamidases (DA) and endopeptidases
(EP) (Sajid et al., 1996; McVeigh et al., 2006a; Husson et al., 2007; Li and Kim, 2008).
SPC disruption in C. elegans and Schmidtea mediterranea causes abnormal distribution of
peptides and deleterious effects on helminth biology. The C. elegans SPC has a unique sequence
at its catalytic domain and may exhibit nematode-specific pharmacology, which makes this
enzyme a possible drug target (McVeigh et al., 2012). Neuropeptide amidation in vertebrates is
facilitated by bifunctional peptidylglycine α-amidating monooxygenase (PAM) with PHM and
PAL domains. In contrast, flatworms encode mono-functional PHM and PAL enzymes on
distinct genes, and C. elegans has multiple copies of mono- and bi- functional enzymes. A PAM
gene mutation in C. elegans causes a lethal phenotype, but experimental evidence of the
essentiality of this enzyme in parasitic nematodes is not yet available. However, the Schistosoma
mansoni PAL and PHM have different structural features and catalytic properties compared to
the respective human host enzyme. Since more than 90% of neuropeptides need to be amidated
to be bioactive and these enzymes are the only ones involved in this process, they have much
appeal as drug target candidates (Mair et al., 2004; Atkinson et al., 2010; McVeigh et al., 2012).
9 2.4.
Neuropeptides
Neuropeptides comprise the most diverse class of signalling molecules used by neurons to signal
to other cells in vertebrates and invertebrates. They can act as direct neurotransmitters, as
modulators of ongoing neurotransmission by other transmitters, or as hormones (Brownlee et al.,
2000; Holmgren and Jensen, 2001; Burbach, 2011). Genes encoding neuropeptides are expressed
in all nerves and neuropeptides can be secreted by any kind of neuron. Classical neuropeptides
are expressed and biosynthesized in neurons, and are stored and released upon demand through a
regulatory secretory route. Overall, neuropeptides mediate or modulate neuronal function by
direct activation of a receptor, which occurs in a relatively slower fashion than observed for other
neurotransmitter receptors mainly because neuropeptides are stored in LCDV. These vesicles are
only recruited to the release site by high intracellular Ca2+ levels, which require prolonged or
intense neuronal stimuli (Burbach, 2011).
The C. elegans genome encodes a wide variety of predicted bioactive peptides (~265) that can be
subdivided in three families according to sequence motifs. These families include the insulin-like
peptides (INS), the FMRF-amide (Phe-Met-Arg-Phe-NH2)-related or -like peptides (FaRPs or
FLPs) and neuropeptide-like proteins (NLPs) (Husson et al., 2007). 40 ins, 42 nlp and 30 flp
precursor genes have been predicted in C. elegans (Li and Kim, 2008). Most INS peptides are
expressed in sensory neurons in almost all life stages and have key roles in metabolism, dauer
formation and development (Husson et al., 2007). NLP peptides do not have a unique sequence
motif, but some of them are homologs of other invertebrate peptides, like allatostatin,
myomodulin, buccalin/drosulfakinin and orcokinin. NLPs have neuronal expression patterns and
are present in other nematodes, including parasites, but are absent in mammals. The biological
functions of most NLPs remain to be studied, but some of them have antimicrobial and
myomodulatory activities (McVeigh et al., 2006b; McVeigh et al., 2008).
The FLP family is the most diverse and structurally conserved family of neuropeptides; many
members have been reported to affect neuromuscular functions. FLPs are consequently the most
studied nematode neuropeptides (Li and Kim, 2008). The nematode flp gene family encodes
more than 70 peptides on 33 precursor genes, three of them parasite-specific. FLPs are 9 -15
10 amino acids in length and, as their name suggests, are similar to the cardioactive tetrapeptide
FMRFamide isolated from the snail Macrocallista nimbosa. The common motif in nematodes is
X/Y-X-R-F-amide, where Y can be any aromatic amino acid, and X a hydrophobic amino acid
such as L, I, V or M (McVeigh et al., 2006a).
RF-amide like peptides are also present in arthropods and other invertebrates. A few peptides
with RFamide C-termini have been reported in vertebrates; however, nematodes FLPs differ
significantly in sequence from their vertebrate counterparts. Nematode FLPs have an
exceptionally broad distribution in the nervous system and play a wide range of physiological
roles, while the vertebrate peptides have a starkly restricted distribution and functionality, being
involved mainly in feeding (Dockray, 2004). Considering the exclusivity of nematodes FLPs and
their functions in neuromuscular control, FLPs and their receptors provide an excellent source of
targets for new anthelmintics that disrupt FLP actions by mimicry or antagonism (Maule et al.,
2002; Mousley et al., 2004; Greenwood et al., 2005; McVeigh et al., 2012).
2.4.1. FMRF-amide like peptides (FLPs) distribution
During the last two decades, with the development of new bioinformatics tools, EST and genome
databases, considerable insight has been gained into the distribution of FLPs and other
neuropeptides across invertebrate phyla (McVeigh et al., 2005; McVeigh et al., 2008; McVeigh
et al., 2009). Using various biochemical and molecular strategies, many FLPs have been
characterized in several members of the order Rhabditida. FLPs from free-living and parasitic
species, including Panagrellus redivivus, A. suum and Onchocerca volvulus (clade III), C.
elegans, Haemonchus contortus and Ancylostoma caninum (clade V), Globodera pallida, G.
rostochiensis, Heterodera glycines, and Meloidogyne arenaria (clade IV), have been purified
and partially characterized.
In addition, studies using RT-PCR, immunocytochemistry, in situ hybridisation (ISH) and
reporter-flp gene constructs (GFP or lacZ reporters) have established that most of the predicted
FLP genes are equally expressed in free-living and parasitic nematodes. They have also been
identified in flatworms and arthropods and there is not much variation in FLP composition in
nematodes across clades or life-styles (McVeigh et al., 2006a; Marks and Maule, 2010). This
11 suggests that the basic biology of peptide-induced signal transduction is evolutionary conserved
among invertebrates (Li, 2006; Marks and Maule, 2010).
As mentioned above, FLPs are widely present in nematode nervous systems, being expressed in
50 -75% of neurons. They occur in all known neuronal subtypes in nematodes, including motor-,
sensory- and inter-neurons; FLP-containing nerves also innervate the pharynx, ovijector and
somatic muscle (Kim and Li, 2004). The methodologies used to detect FLPs are not
neuropeptide-specific due to high similarities between some FLPs and because a single flp gene
can encode multiple different neuropeptides. Additionally, reporter constructs have limitations in
terms of authenticity of expression, potential omission of regulatory elements and difficulties in
promoter identification (Li, 2006). However, each flp gene shows a unique and reproducible
expression pattern.
In C. elegans, the expression of 28 of the 30 candidate flp genes has been confirmed and the
expression pattern of 23, using promoter::gfp reporter constructs, has been established (Kim and
Li, 2004: Marks and Maule, 2010). These genes are expressed in half of the nematode neurons
and each neuron expresses a small subset of flp genes (Li and Kim, 2008). Moreover, some flp
genes have overlapping expression patterns and others are apparently present in non-neuronal
tissues, such as head muscle, pharyngeal muscle, vulva and uterine cells (Kim and Li, 2004;
McVeigh et al., 2006a; Husson et al., 2007). FLP expression patterns in other nematodes show
that there are differences and similarities in FLP profiles between C. elegans and parasites. This
may reflect nervous system plasticity in nematodes, which could facilitate the evolution and
control of behaviors; also the life styles adapted to parasitism that nematodes exhibit despite the
simplicity of their nervous system (Marks and Maule, 2010).
2.4.2. FLP general functions
Physiological studies in A. suum have shown that some FLPs are likely to have physiologically
relevant functions in muscle responses and coordination of behaviours. Using intact A. suum
adult worms or preparations of somatic (body wall) muscle, ovijector muscle and pharyngeal
tissue, it has been demonstrated that nematode and arthropod FLPs induce a diverse array of both
pre- and post-synaptic inhibitory, excitatory and biphasic effects. These physiological effects
12 have been extensively reviewed (see Mousley et al., 2004; McVeigh et al., 2006a; Mousley et al.,
2010) and some are summarized in Table 1.
FLP
gene1
FLP sequence
Common
name2
flp-1
KPNFIRFa
SDPNFLRFa
PF4
PF1
flp-2
flp-3
flp-4
LRGEPIRFa
SPREPIRFa
SPLGTMRFa
ASPSFIRFa
FLP2-1
FLP2-2
FLP3A
FLP4-2
flp-6
KSAYMRFa
FLP6-1/
AF8/PF3
flp-7
SPMERSAMVRFa
FLP7-2
flp-8
KNEFIRFa
FLP8/AF1
flp-9
KPSFVRFa
FLP9
flp-11
AMRNALVRFa
FLP11-1/AF21
NGAPQPFVRFa
FLP11-3/AF22
flp-13
APEASPFIRFa
FLP13-3
flp14
KHEYLRFa
FLP14/AF2
flp-18
GDVPGVLRFa
flp-21
GLGPRPLRFa
Ascaris suum FLP effects
Behavioral
Body wall
Ovijector
effect3
Responses4
muscle
responses5
bwRT2
ovRT1
bwRT1
ovRT1
Abolish body
waves
Ventral coils,
abolish
movement
Abolish body
waves
↓ movement
ovRT3
ovRT2
ovRT1
ovRT1
Pharyngeal
effects6
ND in As
↓ As PP
↓ Ce APF
↑ Ce APF
↓ Ce APF
↑ Ce APF
ovRT1
↓ As PP
↑ Ce APF
ovRT1
ND in Ce APF
bwRT4
ovRT4
ND
ovRT1
↓ As PP
↑ Ce APF
↓ Ce APF
ovRT5
↓ Ce APF
bwRT3 (dorsal)
bwRT1 (ventral)
bwRT4
ovRT1
ND in As
↑ Ce APF
FLP18-6/AF4
Abolish
movement
Flaccid
paralysis
Flaccid
paralysis
Abolish body
waves
↑movement
reduce body
length
↑movement
bwRT3
ovRT2
ND in As
AF9
↓ movement
bwRT3
ovRT2
↓ Ce APF
ovRT1
↓ Ce APF
bwRT1
Table 1. FLP physiological effects in A. suum and C. elegans. 1Gene name in C. elegans, 2. FLP name in A. suum
(AF), in P. redivivus (PF) or C. elegans (FLP) (Li and Kim, 2010), 3Behavioral effects of FLPs injection into
pseudocoelomic cavity (Reinitz et al., 2000; Davis and Stretton, 2001), 4Body wall response types: bwRT1, slow
inhibitory; bwRT2, fast inhibitory; bwRT3, excitatory; bwRT4, biphasic (Maule et al., 1996; Mousley et al., 2005;
McVeigh et al., 2006a), 5Ovijector response types: ovRT1, inhibitory; ovRT2, excitatory; ovRT3, transient
contraction; ovRT4, transient contraction followed by spastic paralysis; ovRT5, relaxation followed by increased
activity (Moffett et al., 2003), 6Pharyngeal effects in A. suum pharyngeal pumping (As PP) and C. elegans
pharyngeal action potential frequency (Ce APF) (Brownlee and Walker, 1999; Rogers et al., 2001; Papaioannou et
al., 2005; Yew et al., 2007).
13 Injection of diverse FLPs directly into the A. suum pseudocoelomic cavity has shown that they
modulate general locomotion, body posture, and head searching activities (Reinitz et al., 2000;
Davis and Stretton, 2001). Additionally, exposure of body wall muscle strips to FLPs induces a
variety of inhibitory and excitatory effects that have been classified as Body Wall Response
Type 1 to 4 (bwRT1- bwRT4). bwRT1 is characterized by a slow and prolonged inhibition,
bwRT2 by a fast and transient inhibition, bwRT3 sustained contraction, and bwRT4 as transient
relaxation followed by sustained contraction (Maule et al., 1996). FLP effects in A. suum at the
neuronal level have been documented using electrophysiological techniques. In total, 21 A. suum
FLPs modify the electrical properties of inhibitory and excitatory motorneurons; they can
depolarize or hyperpolarize motorneuron membranes as well as modify the input resistance
(Davis and Stretton, 1996; Davis and Stretton, 2001).
On the other hand, the effects of ~30 FLPs have been examined in the A. suum reproductive
system, more precisely in the ovijector muscle, which displays rhythmic muscle contraction. The
effects range from slow relaxation and cessation of contractile activity to different contractile
effects, all classified in five Ovijector Response Type categories (ovRT1-ovRT5) (Moffett et al.,
2003). Results suggest that FLPs have a complex role in the modulation of reproductive function,
but the expression of these peptides in the reproductive muscle or in nerves that innervate it is
still unknown and there is not a clear structure-activity correlation within the five ovRTs.
FLP effects in feeding have been assessed by changes in intrapharyngeal pressure and
extracellular recordings of A. suum pharyngeal muscle. A few A. suum peptides inhibit
pharyngeal pumping (Brownlee and Walker, 1999; Yew et al., 2007). FLP effects on pharyngeal
pumping have also been investigated in C. elegans by electrophysiological analysis of
pharyngeal muscle using intracellular and extracellular recordings. 23 C. elegans flp geneproducts modulate feeding by increasing or decreasing pharyngeal pumping frequency. Some
FLPs inhibit the pharynx in similar manner to octopamine, and others increase pharyngeal action
potential frequency as serotonin does (Rogers et al., 2001; Papaioannou et al., 2005). Studies
with C. elegans mutants with phenotypes of defective neurotransmission have shown that some
FLPs have a direct effect on the pharynx; while others mediate their effects indirectly via the
neuronal circuit. Additionally, for some FLPs there is correlation between the flp-gene family,
14 pharyngeal expression and ability to stimulate the pharynx, suggesting they act through receptors
in the pharyngeal muscle. However, pharyngeal expression is not a prerequisite for activity in
pharyngeal muscle and peptides can influence feeding by acting as neurohormones (Papaioannou
et al., 2005). Despite extensive evidence of FLP physiological activity in nematodes, the
mechanisms of action, endogenous targets and functional significance of most FLPs in
nematodes remain largely unknown.
Some flp genes in C. elegans have been overexpressed in transgenesis experiments or inactivated
by gene knockout or RNAi assays to gain insight into their functional roles. Deletion mutants of
eleven flp genes in C. elegans indicated that at least four have behavioral defects and are
involved in locomotion, reproductive behaviour and fat storage (Li, 2006). For example,
inactivation of flp-1 causes hyperactive movement, defects in the timing of egg-laying, reduction
in the number of eggs and fat stores, and insensitivity to high osmolarity (Nelson et al., 1998).
Using RNAi knockdown techniques, behavioral defects, like slow growth, larval arrest and lethal
phenotype, have been observed for another four flp genes. However, most flp-gene loss-offunction mutants show no observable phenotype, even though the encoded FLPs have strong
physiological effects in A. suum preparations. On the other hand, RNAi of three flp homologs in
G. pallida produce profound motor dysfunctions which were not observed in the C. elegans
RNAi studies (Kimber et al., 2007). These discrepancies can be accounted for, in part, by
technique limitations, overlapping FLP functions or functional differences in FLP biology among
species.
FLP physiological effects in other free-living and parasitic helminths have also been studied. For
example, some excitatory A. suum peptides induce contraction of the body wall muscle in
Ascaridia galli, and three different nematode FLPs are active in H. contortus muscle
preparations (Trim et al., 1997; Marks et al., 1999). Flatworm FLPs induce myoexcitatory effects
when applied exogenously to fibres or muscle preparations of various turbellarians,
monogeneans, trematodes and cestodes (Mousley et al., 2010). Overall, some platyhelminth
FLPs induce physiological responses in nematodes and some nematode FLPs are potently
myoexcitatory in flatworms. In addition, three arthropod FLPs inhibit contractility in A. suum
somatic body wall muscle in a similar manner to endogenous FLPs. Other arthropod-specific
15 FLPs are inhibitory in A. suum body and ovijector muscles or induce contraction in muscle fibres
of the free living flatworm Procerodes littoralis (Mousley et al., 2005). Accordingly, FLPs have
the ability to modulate motor events in different species and show inter-phyla activities. This
suggests that there is structural conservation of ligand-recognition features of neuropeptide
receptors across invertebrate phyla even though they are not closely related. It is also possible
that neuropeptide receptors are promiscuous with respect to the peptide ligand; in either case, the
FLP inter-phyla activities make the neuropeptidergic system an excellent source of targets for
exceptionally broad spectrum drugs (Mousley et al., 2004; Mousley et al., 2005).
2.5.
Neuropeptide receptors
Typically, neuropeptide signalling occurs at low peptide concentrations, has a relatively slow
time of onset compared with non-peptide neurotransmitters, and generates long-lasting,
modulatory or metabotropic responses. Consequently, almost all neuropeptides in invertebrates
and vertebrates mediate their effects acting as agonists of G protein-coupled receptors (GPCRs).
GPCRs have seven transmembrane domains and typically interact with ligands in the
extracellular domain and with heterotrimeric GTP binding proteins (G proteins) at intracellular
domains. GPCRs are phylogenetically classified into 5 families named Glutamate, Rhodopsin,
Adhesion, Frizzled/Taste2, and Secretin (Schiöth and Fredriksson, 2005). Neuropeptides
typically activate receptors from the rhodopsin family (class A receptors), which is subdivided
into four main groups and 13 sub-branches based on phylogenetic distance or ligand signature.
Upon ligand binding, a GPCR undergoes conformational changes and releases membraneassociated G-proteins (subunits α, β and γ). The active GPCR acts as a guanine nucleotide
exchange factor (GEF) by inducing GTP (Guanosine triphosphate) exchange in the α subunit and
its consequent dissociation from the βγ dimer; both dissociated α and βγ subunits can elicit
downstream effects. Diverse kinds of α and βγ subunits can amplify the signal through
intracellular pathways, which involve activation of adenylate cyclase (AC), phospholipase C
(PLC), protein kinase A (PKA), protein kinase C (PKC) and inositol 1,4,5-triphosphate (IP3)gated Ca2+ channels, as summarized in Fig. 1. This finally leads to multiple cellular responses,
such as changes in intracellular Ca2+ or cAMP levels, activation of contractile proteins and
16 phosphorylation of other molecules (Bargmann, 1998; Husson et al., 2007; Frooninckx et al.,
2012). The most likely receptors for helminth FLPs are GPCRs, and it is expected that FLPGPCRs signal through the conserved mechanism involving recruitment of a heterotrimeric Gprotein complex (McVeigh et al., 2012).
Figure 1. G protein signalling pathways. Upon ligand (L) binding to the GPCR at the cell membrane (CM) the
receptor-associated Gα-protein exchanges guanosine diphosphate (GDP) to guanosine triphosphate (GTP) and
dissociates from subunits βγ. Depending on the nature of the Gα subunit, different signalling pathways can be
induced. Gαi or Gαs release results in inhibition or activation of adenylyl cyclase (AC), respectively, leading to
changes in intracellular cAMP levels and inhibition/activation of protein kinase A (PKA). Gαq can directly stimulate
phospholipase Cβ (PLCβ) to induce release of intracellular Ca2+ from the endoplasmic reticulum (ER). Release of
Gβγ subunits has the same effect on PLCβ, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into
inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) to activate protein kinase C (PKC) (Frooninckx et al.,
2012).
Many G proteins have been identified in C. elegans, including 21 different Gα subunits and 2 βγ
dimmers. The C. elegans Gα family includes orthologs of each of the mammalian Gα subunits:
Gs (gsa-1), Gi/o (goa-1), Gq (egl-30) and G12 (gpa-12), as well as 17 C. elegans-specific α
subunits with no clear homologs in mammals (Bastiani and Mendel, 2006). Additionally, Gprotein targets in classical signaling pathways are present in C. elegans, including PLC (egl-8),
AC (acy-1), IP3 (itr-1), and UNC-13 which regulates synaptic vesicle release of ACh (Husson et
al., 2007; Frooninckx et al., 2012). Loss-of-function mutations in these signalling molecules
produce aberrant phenotypes in C. elegans, and physiological studies in A. suum show changes
17 in cAMP levels caused by exposure to FLPs. However, there is no evidence of pharmacological
or structural differences between mammalian and nematode G proteins and their downstream
secondary messengers; therefore, they are not obvious targets for parasite-specific intervention
(Geary, 2010).
The search for neuropeptide receptors has been focused on GPCRs in C. elegans and parasitic
helminths. To date, 23 GPCRs have been associated with neuropeptides, including 12 C. elegans
FLP-GPCR systems, one FLP-GPCR in a parasitic nematode and one from a planarian flatworm
have been predicted (Omar et al., 2007; Frooninckx et al., 2012; Atkinson et al., 2013).
However, not all neuropeptides signal through GPCRs; indeed, the first FLP receptor identified
in invertebrates was a Na+ channel (NaCh) from the snail Helix aspersa. This receptor is not
closely related to the epithelial NaChs and degenerins of C. elegans. It is amiloride sensitive and
gated by FMRFa and FLRFa peptides (Lingueglia et al., 1995). In addition, physiological
evidence from A. suum suggests that the PF4 peptide (KPNFIRFa) may directly gate an ion
channel. PF4 causes a rapid relaxation of A. suum body wall muscle, hyperpolarizes the somatic
muscle membrane and increases its input conductance in a Cl--dependent manner. PF4 acts with
the same speed as GABA in muscle tissue as well as at the membrane, but in an independent
manner (Holden-Dye et al., 1997; Purcell et al., 2002). However, no Cl- ion channel has been
identified as the receptor for PF4.
2.5.1. Neuropeptide GPCR identification
Using the Lymnaea stagnalis lymnokinin GPCR as a search query in the C. elegans genome
database, around 60 GPCRs were predicted as putative receptors for small-molecule transmitters
and neuropeptides. They are part of the Rhodopsin family and can be classified in subgroups
based on sequence similarities to vertebrate and insect GPCRs (Keating et al., 2003). Recently,
the list has been expanded to 125 putative neuropeptide GPCRs using specific motifs from
peptide-GPCRs for which ligands are known in a MEME/MAST analysis (Multiple Expectation
Maximization for Motif Elicitation/Motif Alignment and Search Tool) (Janssen et al., 2010). The
predicted receptors appear to be less closely related to their vertebrate counterparts and most C.
elegans neuropeptide-GPCRs are worm-specific, which agree with the low conservation of worm
18 neuropeptides (Bargmann, 1998; Frooninckx et al., 2012). Unfortunately, not much information
about neuropeptide GPCRs in parasitic nematodes is available, but since FLPs are conserved
across the phylum, similar conservation at the receptor level is expected. This assessment
validates the use of C. elegans for neuropeptide receptor characterization (McVeigh et al., 2012).
Functional genomics studies in C. elegans have shown that mutations in some peptide GPCRs
induce deleterious phenotypes, such as lethality, reduced lifespan, uncoordinated motility or
defective reproduction (McVeigh et al., 2012). RNAi assays have also identified GPCRs that
yield deleterious phenotypes. However, RNAi knockdown assays do not always reproduce
phenotypes obtained by genetic modification, and some GPCRs are resistant to disruption by
RNAi (Keating et al., 2003). In total, 21 putative neuropeptide GPCRs yield deleterious
phenotypes, following knockout or RNAi analysis, which resemble to those phenotypes induced
by
anthelmintic
exposure.
This
suggests
that
neuropeptide
GPCRs
are
legitimate
chemotherapeutic targets and that receptor antagonists may exhibit therapeutically useful
pharmacology for at least some neuropeptide GPCRs. On the other hand, the profound
physiological effects observed for FLPs indicate that agonists will also be of value for
chemotherapy (McVeigh et al., 2012). In either case, the identification of endogenous ligands for
nematode peptidergic GPCRs (deorphanization) is the first step needed to enable functional
characterization of these GPCRs and to establish their utility as drug targets.
2.5.2. FLP-GPCR deorphanization
The typical approach to deorphanize or match GPCRs with their cognate ligand is by reverse
pharmacology, in which candidate receptors are expressed in a heterologous system, such as
mammalian cells or Xenopus laevis oocytes. The heterologously expressed receptor is used as
bait to be tested with pools of synthetic FLPs in a high-throughput format using as a read out
different elements of the GPCR-signaling cascade. If activation of receptor and ligand titration is
observed at physiological concentrations with a particular FLP, the match is considered positive
(Husson et al., 2007). Successful ligand-identification experiments have used receptors
expressed together with chimerical G proteins in Chinese Hamster Ovary (CHO) cells; receptor
activation is followed by membrane binding of a non-hydrolysable form of GTP (GTPγS)
19 (Kubiak et al., 2003a; Kubiak et al., 2003b; Lowery et al., 2003; Kubiak et al., 2007), or by
increases in intracellular [Ca2+] measured in fluorescence assays (Mertens et al., 2004; Mertens
et al., 2005a; Mertens et al., 2005b; Mertens et al., 2006).
With similar approaches, 12 C. elegans GPCRs (including some isoforms) have been matched
with FLPs (Table 2). For instance, receptor NPR-1, a homolog to the mammalian neuropeptide
Y receptor, was matched with GLGPRPLRFa (AF9/FLP-21) by screening >200 peptides in
pools using a GTPγS readout in mammalian cells grown at 28°C. FLP-21 has differential affinity
for two NPR-1 isoforms which differ in one amino acid at position 215 (F or V) (Kubiak et al.,
2003a). This ligand-receptor association was confirmed by NPR-1expression in X. laevis oocytes
and ectopically in C. elegans pharynx; both receptor variants were activated by FLP-21 and the
215V form by FLP-18 peptides (-PGVLRFa) (Rogers et al., 2003). Mutations in the NPR-1
receptor affect aggregation behavior: animals aggregate during feeding and accumulate at the
edge of the bacteria in plates. Animals carrying a loss-of-function (lf) mutation in the precursor
flp-21 display similar aggregation during feeding, suggesting that this ligand and the receptor are
in the same signalling cascade. However, the phenotype of flp-21(lf) is mild compared to the
npr-1 (lf), probably because NPR-1 can be activated by FLP-18 peptides in the absence of FLP21 and the 215V allele is involved in solitary feeding (Li and Kim, 2008).
As shown in Table 2, some GPCRs have exquisite ligand specificity, with a physiological
binding affinity in the range of 1 – 10 nM. Others have affinity in the range of ≥ 1 μM and bind
multiple ligands encoded by one gene or different genes. The similarities in FLP sequences, their
overlapping expression patterns and the diversity in tissue responses caused by FLPs, make it
theoretically possible that one receptor can be activated by more than one FLP and that one FLP
may act on several receptors (Li and Kim, 2008). However, some matched receptors expressed in
heterologous systems can distinguish among neuropeptides with similar C-termini or that differ
in a single amino acid (Kubiak et al., 2003b; Mertens et al., 2004). This is the case for FLP-21
(GLGPRPLRFa) and FLP-15 (GGPQGPLRFa), which do not activate the same receptor
despite highly similar sequences. This evidence supports the complexity of the neuropeptidergic
system and suggests that organism-level studies are needed to fully characterize ligand-receptor
pairs and functionally confirm receptor promiscuity.
20 On the other hand, for some matched receptors, elements of downstream signalling pathways in
heterologous systems have been identified. NPR-1, upon activation by FLP-21, causes the
exchange of GDP for GTP on the Gαi/0 subunit and inhibits AC as the main signalling pathway
for both receptor variants expressed in mammalian cells (Kubiak et al., 2003a). In contrast, the
ligand/receptor system FLP-18/NPR-5 activates the Gαq subunit and is involved in intracellular
Ca2+ release, probably via PLC (Kubiak et al., 2007). Specific Gα subunits have been implicated
in signalling pathways of other FLPs even though their receptors have not been identified. This is
the case for KHEYLRFa (AF2/FLP-14), which induces GDP-GTP exchange and GTP hydrolysis
by a Gα subunit in body wall muscle membranes, consistent with direct activation of a GPCR by
AF2 (Kubiak et al., 2003c).
Using high throughput heterologous expression, more than 60 C. elegans GPCRs have been
tested (Lowery et al., 2003), but overall the reverse pharmacology approach has produced
relatively few matches. Despite the many advantages of using C. elegans as a model and large
investment by several laboratories, at least 70% of putative neuropeptide GPCRs in this
organism remain unpaired (McVeigh et al., 2006). The reverse pharmacology approach
successfully deorphanized a high number of mammalians and Drosophila GPCRs at the
beginning of this century (Larsen et al., 2001; Wise et al., 2004; Lecca and Abbracchio, 2008).
But the deorphanization rate has slowed since these early success, perhaps due to inherent
problems encountered in the heterologous expression of invertebrate GPCRs, such as poor
trafficking to the cell surface, lack of ligand access to the binding site, poor coupling with
signalling proteins, and the need for additional receptor-interacting proteins in the heterologous
system (Levoye and Jockers, 2008; Dunham and Hall, 2009). It may be the case that all the
‘easy’ nematode peptidergic GPCRs were readily matched, and that further progress in this area
will be difficult.
An RNAi-mediated GPCR deorphanization approach has recently been proposed. The strategy
consisted in identifying changes in GPCR secondary messengers in the native cell or membrane
environment upon ligand exposure, including neuropeptides. RNAi of selected candidate GPCRs
is monitored to detect reductions in response to ligand exposure caused by the target receptor
knockdown (Zamanian et al., 2012). In another example, ligand-receptor RNAi-phenotype
21 matching enabled the G. pallida FLP-32 GPCR to be deorphanized, allowing the first functional
characterization of a parasitic nematode FLP-GPCR (Atkinson et al., 2013). Although these
strategies can overcome some of the limitations of reverse pharmacology, RNAi methods have
their own limitations, such as resistance to RNAi in some tissues, lack of a detectable receptor
knockdown phenotype, and the need for large amounts of membrane preparations, which is often
challenging for parasitic helminths. New strategies for matching are still needed.
Gene name
C26F1.6
Receptor
name
FRPR-3
Y59H11AL.1a
NPR-22a
T19F4.1a
FRPR-18a
T19F4.1b
FRPR-18b
C10C6.2
NPR-3
C25G6.5
NPR-11
C39E6.6 (V)
NPR-1
C39E6.6 (F)
NPR-1
C16D6.2
NPR-4
Y58G8A.4a
NPR-5a
Y58G8A.4b
C53C7.1a
NPR-5b
NPR-10a
Most potent FLP
FLP7-1:
TPMQRSSMVRFa
FLP7-2:
SPMERSAMVRFa
FLP2-1 :
SPREPIRFa
FLP2-1:
SPREPIRFa
FLP15-1:
RGPSGPLRFa
FLP1,-4,-5,-14,-18
FLP-21:
GLGPRPLRFa
FLP18-2:
EMPGVLRFa
FLP-21:
GLGPRPLRFa
FLP4-2:
ASPSFIRFa
FLP18-2:
SVPGVLRFa
AF3:
AVPGVLRFa
FLP18-6:
GDVPGVLRFa
FLP18-2:
SVPGVLRFa
AF3:
AVPGVLRFa
FLP18-6:
GDVPGVLRFa
FLP3:
-(L/F)GTMRFa
Expression
system1
CHO/FA
HEK293/FA
HEK293/FA
1.1 ± 0.1 μM
CHO/FA
53.1 ± 7.7 nM
CHO/FA
54.4 ± 6.2 nM
CHO/GTPγS
162 nM
(Mertens et al.,
2005a)
(Mertens et al.,
2005a)
(Kubiak et al., 2003b)
CHO; HEK293/FA
HEK293/GTPγS
CHO/GTPγS
Xo/VCA
Xo/VCA
1-8 μM
(Lowery et al., 2003)
2.5 nM/-107.8
μA2
(Kubiak et al., 2003a;
Rogers et al., 2003)
-32.2 μA2
102.5 nM/
104.6 μA2
5-80nM
(Kubiak et al., 2003a;
Rogers et al., 2003)
(Lowery et al., 2003)
CHO/GTPγS
Xo/VCA
CHO; HEK293/FA
HEK293/GTPγS
CHO/FA
EC50
1.0 ± 0.2 μM
Reference
(Mertens et al., 2004;
Mertens et al., 2005b)
(Mertens et al., 2006)
(Kubiak et al., 2007)
14.1 nM
7.6 nM
13.5 nM
CHO/FA
(Kubiak et al., 2007)
43.3 nM
13.1 nM
CHO; HEK293/FA
HEK293/GTPγS
37.2 nM
60-300 nM
(Lowery et al., 2003)
Table 2. Deorphanized FLP-GPCRs by high-throughput screening. 1CHO: Chinese hamster ovary cells; FA:
Calcium mobilization-fluorescence assay; HEK293: Human embryonic kidney cells; GTPγS: binding assays with
non-hydrolysable form of GTP; Xo: Xenopus laevis oocytes; VCA: Voltage clamp assays. 2Inward current in VCA
after activation of co-expressed G-protein-regulated inward-rectifier K+ channels (GIRKs) (Rogers et al., 2003).
22 2.6.
FLP-GPCRs as drug targets
The FLP neuropeptidergic system is an attractive source of new anthelmintic targets for many
reasons. First, as mentioned before, FLPs are involved in a variety of neuromuscular functions
that are essential for parasite viability in the host. Second, these peptides are found almost
exclusively in invertebrates and related peptides appear to be of limited importance in mammals,
suggesting a high probability that new compounds would have selective toxicity for parasites.
Third, FLPs are ubiquitous in all target invertebrates, including parasitic nematodes, flatworms
and arthropods. Although some are unique to a particular species, there is broad conservation of
FLP sequences among a phylum. Finally, FLPs are promiscuous and have shown cross-phylum
activity, probably by binding similar receptors in different phyla. This suggests that drugs against
the FLP system could have low toxicity and remarkably broad spectrum activity. In addition
there is low chance of cross-resistance since no known drug targets the FLP system (Maule et al.,
2002; McVeigh et al., 2012).
However, FLPs themselves are of limited therapeutic value and cannot be used as drugs. FLPs,
like other peptides, will not be orally available (they are degraded in the stomach and lack
transport mechanisms) and will be quickly degraded in blood by proteolytic digestion. Due to
their generally hydrophilic nature, they will diffuse poorly across the gut and nematode surfaces.
These characteristics, together with their high cost of production and purification, make FLPs
poor drug candidates. Instead, small, non-peptide molecules that mimic or block FLP effects at
the receptor level are needed, much like morphine and the enkephalin peptides (Hökfelt et al.,
2003; Janecka et al., 2004; Brain and Cox, 2006). Consequently, FLP-GPCRs, peptide-gated ion
channels or other neuropeptide GPCRs can only serve as targets for new drug discovery.
Although no known anthelmintics target GPCRs in parasites, they are highly druggable. Some
40% of the most successful drugs used in human medicine act on GPCRs, and there is much
experience with these drugs (Wilson et al., 1998; Jacoby et al., 2006; Lecca and Abbracchio,
2008). It is also known that FLP-GPCRs can be expressed in heterologous system for highthroughput screening of chemical libraries. In addition, FLP-GPCRs have no close homologs in
vertebrates and the broad distribution and expression of their FLP ligands suggests that FLP23 GPCRs are present in key parasites and key life stage(s) and function in therapeutically sensitive
target tissues, like the neuromuscular system. These factors support the potential of FLP-GPCRs
as targets for the discovery of very broad-spectrum anthelmintics (Geary, 2010; McVeigh et al.,
2012).
Discovery efforts are limited by the fact that many aspects of the nematode FLP system are
unknown; basic research is needed into the functional relevance of FLP-GPCRs. For instance, in
order to propose a FLP-GPCR as a rational drug target, its cognate ligand/ligands must be
known, along with the signalling pathway, the kind of responses that the receptor generates, the
exclusivity of its signalling and its importance in nematode physiology, viability and behaviour,
as well as maintenance of parasitic species in their hosts. Considering the complexity of the FLP
system and the possible functional redundancy of peptides and receptors, non-peptide ligands
must be promiscuous acting on multiple FLP-GPCRs to successfully have as much of an impact
in parasite biology as current anthelmintics do, and deter the evolution and spread of receptormediated resistance mechanisms (Maule et al., 2002; Greenwood et al., 2005).
In terms of GPCR deorphanization, high-throughput screening methodologies have not been
successful in pairing most candidate FLP-GPCRs with their cognate ligand/ligands. Moreover,
the most profoundly active FLPs in physiological studies remain ligands for orphan receptors
and not enough information about the physiological roles of the deorphanized FLP-GPCRs has
been gained; therefore, they cannot be confidently targeted in mechanism-based screens for
novel anthelmintics. Considering the limitations of heterologous expression systems for
deorphanization, in situ organism-based strategies provide an attractive alternative for matching
and receptor characterization. Since C. elegans remains the predominant source of information
about nematode neuropeptidergic systems and there is strong conservation of FLPs and receptors
across the phylum, is valid to do in situ deorphanization in C. elegans. In addition, using C.
elegans brings diverse technical advantages, including easy cultivation, a fully sequenced
genome, a well-documented wiring system, ease of genetic manipulation and thousands of
mutant strains and genetic libraries, tools that are not yet available for parasitic nematodes.
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As a free-living nematode, C. elegans deals with many types of xenobiotics commonly found in
the soil environment. Its exoskeleton, the cuticle, acts as a permeability barrier to protect it from
stressors and insults found in its environment. Some molecules, including some drugs, are able to
cross the cuticle and reach their targets in intact C. elegans specimens. For example,
anthelmintics like levamisole, pyrantel and bephenium induce contractile paralysis in direct C.
elegans bioassays. Other molecules, like the novel anthelmintic nAChR antagonist derquantel (2desoxoparaherquamide, 2-DOPH) and neuropeptides, do not have evident effects in intact
specimens. To determinate whether the cuticle is a barrier to the entry of these molecules, we
adapted a cut-worm model in which worms are bisected between the anterior end and the mildpoint, preserving motor activity. In the following chapter, I focus on the use of the cut-worm
model to investigate the activity of 2-DOPH in C. elegans in a proof-of-concept study to
investigate whether the barrier properties of the C. elegans cuticle can be overcome in a lowthroughput bioassay. We also used the model to evaluate the effect of other nAChR antagonists
and agonists, including the new amino-acetonitrile derivative monepantel. Results from this
study validate the use of the cut-worm model to study impermeant neuroactive peptides in situ
that will be discussed in Chapter III.
30 Chapter II. Manuscript I.
Activity of the novel nicotinic anthelmintics in cut preparations of Caenorhabditis elegans
Elizabeth Ruiz-Lancheros, Charles Viau, Tita N. Walter, Abdel Francis, Timothy G. Geary
Institute of Parasitology, McGill University,
21111 Lakeshore Road, Ste-Anne-de-Bellevue QC Canada H9X 3V9
Published in: International Journal for Parasitology, 41: 455-461
(2011)
31 Abstract
The free-living nematode Caenorhabditis elegans is a useful model for studying the
pharmacology of anthelmintics. Currently approved anthelmintics have various mechanisms of
action, including activity at nematode nicotinic acetylcholine receptors (nAChRs). Classical
anthelmintic agonists of these receptors (nicotine, levamisole, pyrantel and bephenium) caused
intact specimens of C. elegans to undergo contracted paralysis. The nAChR antagonist
mecamylamine paralyzed intact worms and blocked the actions of the agonists. The time to onset
of effects of these drugs was enhanced when worms bisected between the mid- and anterior
portions
were
tested.
The
novel
anthelmintic
nAChR
antagonist
derquantel
(2-
desoxoparaherquamide, 2-DOPH) was weakly active in intact specimens of C. elegans at
concentrations of 50 μM over several days. No antagonism of the nAChR agonists was observed
with this drug in intact worms. However, derquantel had direct and marked effects on motility in
cut worms and blocked the effects of nAChR agonists in this preparation. A representative of the
new amino-acetonitrile derivative (AAD) class of nAChR agonists was not antagonized by
derquantel in cut C. elegans, suggesting that these two anthelmintics may not demonstrate
unfavourable drug-drug interactions at the receptor level if used to treat livestock infected with
parasitic nematodes. The permeability properties of the C. elegans cuticle may be more
restrictive than those of adult parasites, calling into question primary anthelmintic screening
strategies that rely on this model organism.
32 1.
Introduction
Several classes of anthelmintics are in use in veterinary and human medicine for control of
nematodiases (see Lacey, 1988; Martin et al., 1997; Holden-Dye and Walker, 2007 for review),
including the γ-aminobutyric acid (GABA) agonist piperazine, inhibitors of tubulin
polymerization in the benzimidazole class, macrocyclic lactones, which open glutamate-gated Clchannels, emodepside, a cyclodepsipeptide thought to open a potassium channel,
diethylcarbamazine, nitazoxanide, neither with a known mechanism of action, and agonists of
nicotinic acetylcholine receptors (nAChRs) including pyrantel, levamisole and monepantel, a
new drug in the amino-acetonitrile derivatives (AAD) class (Kaminsky et al., 2008). As with all
chemotherapeutic agents, resistance to anthelmintics is a continuing concern (Gilleard and
Beech, 2007). Derquantel (2-desoxoparaherquamide; 2-DOPH), a novel nAChR antagonist (Lee
et al., 2002), is effective against nematodes which are resistant to other anthelmintics (Zinser et
al., 2002). This compound is of interest compared with its parent compound, paraherquamide, in
part because of its apparently lower toxicity in some mammalian species (Lee et al., 2002).
Much can be learned about anthelmintics in experiments using the free-living nematode
Caenorhabditis elegans (Holden-Dye and Walker, 2007). This worm can be easily maintained in
culture in the laboratory, especially compared with parasitic worms, which have complex lifecycles. It also has a sequenced genome and an impressive molecular toolkit for functional
genomics experiments, and genetic screens can be easily carried out in it to decipher mechanisms
of action (Holden-Dye and Walker, 2007; Kaminsky et al., 2008). However, it is not a perfect
model for parasitic species (Geary and Thompson, 2001; Holden-Dye and Walker, 2007). Only
about 70-80% of genes in parasitic nematodes have obvious homologs in C. elegans (Geary and
Thompson, 2001; Ghedin et al., 2007) and their intermediary metabolisms differ in some
respects (Geary and Thompson, 2001). Caenorhabditis elegans lives in the soil and is more
adapted to deal with stresses and xenobiotics common to soil environments than are adult stages
of parasitic nematodes, which are adapted for existence in different compartments within a host
and are the primary targets for anthelmintic chemotherapy (Geary and Thompson, 2001). It can
be argued that C. elegans is better equipped to deal with xenobiotic insults found in its
environment (potential drugs) than are adult stages of parasitic species in this phylum; free-living
33 larval stages of trichostrongylid nematodes, which also exist in external environments, may also
be more resistant to xenobiotic stresses (and are generally much less sensitive to anthelmintics
than are adults; Folz et al., 1987). In this context, it is interesting to note that the novel
anthelmintic agent 2-DOPH has not been reported to act on C. elegans despite the fact that it is
quite potent against many species of parasitic nematodes in culture and in hosts (Lee et al.,
2002). Although paraherquamide has been reported to be active in C. elegans, the action is
difficult to discern and is not lethal (T.G. Geary, unpublished observations), although this
chemical class has a membrane binding site in C. elegans (Schaeffer et al., 1992).
The exoskeleton of nematodes, the cuticle, is a permeability barrier to xenobiotics (Ho et al.,
1990; Thompson et al., 1993; Ho et al., 1994; Thompson and Geary, 2003). To determine
whether the cuticle of C. elegans is a barrier to the entry of 2-DOPH, thus accounting for its very
weak activity against this organism in bioassays, we adapted the cut-worm model introduced by
Lewis et al. (1980) to analyze the pharmacology of this novel nAChR ligand. In this model,
worms are cut at approximately one-third of their length between the anterior end and mid-point.
Responses to nicotinic ligands occurred much more quickly and at lower concentrations in cut
versus intact specimens (Lewis et al., 1980). This model is thus well-suited to test the hypothesis
that low sensitivity to paraherquamides in C. elegans compared with parasitic nematodes is due
to differences in cuticle permeability, as opposed to differences in receptor abundance or affinity.
In the current work, responses to nAChR agonists were used to monitor the effects of 2-DOPH
and mecamylamine, a known nAChR antagonist. The model was also used to evaluate possible
receptor-level interactions between AAD and paraherquamide-class anthelmintics, which could
influence their deployment in the field.
2.
Material and methods
2.1.
Cultures of C. elegans
Caenorhabditis elegans N2 strain cultures were grown in Petri dishes containing NGM
(Nematode Growth Medium) medium seeded with Escherichia coli strain OP50 and were
synchronized by standard methods (Wood, 1988). The strain was obtained from the
Caenorhabditis Genetic Center (University of Minnesota, Minneapolis MN, USA).
34 2.2.
Drug solutions
Drug solutions were prepared in artificial perienteric fluid (APF), composed of 67 mM NaCl, 67
mM NaCH3COOH, 3 mM KCl, 15.7 mM MgCl2, 3 mM CaCl2 and 5 mM Tris (Parri et al.,
1991). Glacial acetic acid was added to reach pH 7.6. For stock solutions (all 10-2 M), the
following specifications were followed. Nicotine in liquid form (nicotine hemisulfate) was
directly diluted into APF. Pyrantel tartrate was dissolved in double distilled water (ddH2O) with
heating at 45°C or in DMSO. Levamisole HCl and mecamylamine HCl were also dissolved in
ddH2O. Bephenium hydroxynaphthoate was dissolved in 100% ethanol with heating at 45°C. All
of these compounds were obtained from the Sigma Chemical Co., St. Louis, MO, USA. 2-DOPH
stock solution was prepared from powdered drug, kindly provided by Pfizer Animal Health
(Kalamazoo, MI, USA), and dissolved in methanol. AAD 1470 (Kaminsky et al., 2008), kindly
donated by Novartis Animal Health, Basel, Switzerland, was dissolved in DMSO.
2.3.
Worm assays
Cut worm assays were adapted from published protocols (Lewis et al., 1980), using synchronized
L4 – young adult stage C. elegans N2 cultures. A plain glass microscope slide was divided into
two equal squares with Vaseline (Scott Chemical Canada) using a fine paint brush. The thickness
of the Vaseline barriers was maximized to optimize depth of the liquid layer and retard
evaporation during the assay. APF (100 µl) was added to the first square. Five C. elegans young
adults were transferred to the APF solution. Manipulations were performed under a dissecting
light microscope at ~2.5 V, which did not appear to damage C. elegans and minimized
evaporation.
A thin layer of silicone grease was applied to a surgical blade to facilitate cutting. C. elegans
were severed between the middle and anterior portions, approximately two-thirds from the
anterior end. Only cleanly cut specimens were used; worms were rejected if filaments extended
from the anterior portion. APF (100 µl) or drug-APF solution was added to the second square of
the microscope slide. With a hair held by the root, the anterior portion of the C. elegans was
transferred to the second square. The assay timer was started when the specimen was introduced
35 into this solution. Four C. elegans specimens were sequentially transferred to the second square,
registering the time at which each was added to the second square (time introduced = ti).
For each treatment, the behaviour of 10 – 20 cut worms was assessed, using as a treatment
control cut worms maintained in APF supplemented with the appropriate solvent. To ensure C.
elegans were paralyzed and not just resting, the ends of non-moving specimens were tapped with
a hair. If the worm curled easily, it was considered to not be paralyzed. If the worm curled
weakly or not at all, it was rated as paralyzed. The time at which a specimen was considered
paralyzed was registered (time paralyzed = tp). Therefore, time to paralysis = tp-ti. The mean time
to paralysis was calculated for groups and used for comparisons.
Individual drugs and their mixtures were tested randomly on different days by more than one
person in a blinded format to avoid bias and ensure reproducibility and consistency. Experiments
were repeated in three to six independent replicates. The same procedure, minus the cutting step,
was followed for short-term assays with intact worms.
Data analysis was performed using GraphPad software (GraphPad, San Diego, CA, USA).
Paralysis was analyzed as the percentage of the population that were paralyzed at specific times
during the study period. Curves were fitted to the data using non-linear regression analysis.
Results for a single set of experiments are shown when population analysis was done. For these
experiments, at least three independent replicates were performed. Time to paralysis in control
incubations was influenced by difficult to control variations in laboratory temperature and
humidity. Thus, we present results for a single population in Figures 1, 3, 4 and 5, noting that
similar drug effects were observed in three replicate experiments. S.E.M. statistics are shown in
Figure 2 and 6 when mean of time-until-paralysis was used as the parameter of drug effects.
3.
Results
3.1.
Agonist concentration-response studies
To confirm that nAChR pharmacology can be studied in cut C. elegans, nicotine, pyrantel,
levamisole and bephenium were tested on cut and uncut worms. Concentration-response curves
for these drugs in cut specimens mimicked those reported previously (Lewis et al., 1980);
36 examples of data for pyrantel are shown in Fig. 1. Time-to-paralysis decreased with increasing
agonist concentration. Bephenium was the least potent (not shown). Potency of these drugs in the
short time-frame of these incubations was increased when C. elegans specimens were bisected
compared with intact worms; data for pyrantel again illustrate this effect (Fig. 2). The duration of
observation for data analysis in these experiments was limited to no more than 90 min for intact
worms, as evaporation from the test wells compromised motility of drug-free controls by that
time. Experiments using cut worms terminated by 30 min, at which time control worms lost
viability. Worms exposed to solvents alone at the concentrations used for these drugs did not
differ in response compared with worms exposed to medium only in either case (not shown).
Figure 1. Effects of pyrantel on cut Caenorhabditis elegans. Each point represents the percentage of 10-12 worms
which were non-motile at the specified time and drug concentration. Population analysis for a single set of
experiments is shown. However similar results were obtained in three independent replicates rated by two different
observers.
37 Figure 2. Comparative effects of pyrantel on cut and intact Caenorhabditis elegans. Mean time until paralysis
is reported for replicates of 10-12 specimens at each concentration. Error bars represent S.D; similar results were
obtained in an independent replicate
3.2.
Cut worm assay with mecamylamine as an nAChR antagonist
Mecamylamine had a paralytic effect at high concentrations. The time at which 50% of the
worms were paralyzed in the absence of drug was approximately 30 min, while for
mecamylamine (10-4 M), it was ~ 20 min (not shown). The extent of the difference was
consistent in independent replicates, although variations in ambient humidity and temperature led
to variability in the time to paralysis in the control samples. The drug was inactive in this assay
at 10-5 M and had no detectable activity in uncut worms in the limited duration of exposure used
in these experiments (data not shown). Mecamylamine was used at 10-4 M in subsequent assays.
Mecamylamine antagonized the paralytic effects of nAChR agonists in the cut worm assays (Fig.
3 A-D) as evidenced by a shift to the right in the time-to-paralysis curves for pyrantel and
levamisole, in the presence of 10
-4
M mecamylamine at low agonist concentrations. The short
duration of these experiments precluded pre-incubation of the cut preparations with
mecamylamine, which may have prevented us from detecting antagonism at higher agonist
concentrations. A shift to the right for 10-5 M nicotine and bephenium was observed with 10-4 M
mecamylamine. The variation inherent in the experimental preparation prevents a conclusion that
mecamylamine antagonized these agonists with different affinity.
38 Figure 3. Antagonism by mecamylamine. A shift to the right in the time-to-paralysis curve was observed for
different nicotinic acetylcholine receptor (nAChR) agonists in the presence of mecamylamine (Mec.). (A) Pyrantel;
(B) levamisole; (C) bephenium; (D) nicotine. Each point represents the percentage of 10-12 specimens which had
lost motility. The concentration of mecamylamine used did not cause paralysis on its own. Population analysis for a
single set of experiments is shown in each case. However similar results were obtained in three independent
replicates rated by two different observers.
3.3.
Effects of 2-DOPH on C. elegans
2-DOPH, at the highest concentration tested (10-4 M), was indistinguishable from control in
time-to-paralysis in intact specimens. Although 2-DOPH (10-4 M) had a slight antagonistic effect
on levamisole (10-5 M) in short-term assays on whole C. elegans, this effect was not statistically
39 significant (not shown), nor was it consistently observed in additional replicates. However, at 24
h in 12-well plates, 2-DOPH (≥ 20 μM) caused curling behavior that could reflect muscle
contraction, although the worms remained motile and viable (not shown). In contrast, 2-DOPH
rapidly paralyzed cut preparations of C. elegans at concentrations as low as 10-5 M (Fig. 4).
Figure 4. 2-Desoxoparaherquamide (2-DOPH) paralyzes cut specimens of Caenorhabditis elegans. Each point
represents the percentage of 10-12 worms which had lost motility. Population analysis for a single set of
experiments is shown. However similar results were obtained in three independent replicates rated by two different
observers.
3.4.
2-DOPH is an nAChR antagonist
Like mecamylamine, 2-DOPH antagonized nAChR agonists in cut C. elegans preparations (Fig.
5 A-D). 2-DOPH appeared to be a more potent antagonist than mecamylamine. Analysing the
time at which 50% of the population became paralyzed in the presence of agonist alone
compared with agonist + 2-DOPH revealed that 2-DOPH is a more profound antagonist of
bephenium and pyrantel than of levamisole. The antagonism of nicotine was only evident at 105
M, making it difficult to define its sensitivity to 2-DOPH antagonism relative to the other drugs.
40 Figure 5. 2-Desoxoparaherquamide (2-DOPH) antagonizes the actions of nicotinic acetylcholine receptor
(nAChR) agonists on cut specimens of Caenorhabditis elegans. Each point represents the percentage of 10-12
specimens which lacked motility. (A) Pyrantel; (B) levamisole; (C) bephenium; (D) nicotine. Population analysis for
a single set of experiments is shown. However similar results were obtained in three independent replicates rated by
two different observers.
Interaction of 2-DOPH and AAD 1470 in cut worm assays
The AAD class is potently active against intact C. elegans, in which it acts on a novel type of
nAChR (Kaminsky et al., 2008). To assess whether 2-DOPH is an antagonist at these receptors,
we studied the interaction of these drugs in cut preparations of C. elegans. In these experiments,
approximately 30 specimens were incubated with the drugs alone or in combination and the
mean time until paralysis was compared between treatments. 2-DOPH concentrations as high as
10-4M failed to delay the onset of paralysis in cut C. elegans caused by low concentrations of the
41 agonist AAD 1470 (Fig. 6). The interaction of the two drugs varied from no change in time until
paralysis compared with the more rapidly acting AAD alone, to a slight additive effect. These
experiments were not designed to detect additive or synergistic effects of the two drugs, but
clearly show absence of antagonism. Interestingly, mecamylamine (10-4 M) was also not an
antagonist of AAD 1470; the two drugs tended to be additive, although the difference compared
with AAD 1470 alone was not consistently observed (Fig. 6).
Figure 6. 2-Desoxoparaherquamide (2-DOPH) and amino-acetonitrile derivative (AAD) 1470 do not interact
in cut specimens of Caenorhabditis elegans. Bars show the mean time until paralysis of 10-12 worms incubated in
AAD 1470 (10 or 100 μM), alone or plus 2-DOPH (10 or 100 μM) or mecamylamine (Mec. 100 μM). Similar
results were seen in four independent replicates. Overall, no consistent differences in time until paralysis were
observed for AAD 1470 plus any antagonist at any concentration.
4.
Discussion
2-DOPH is an nAChR antagonist at pharmacologically relevant concentrations in parasitic
nematodes (Lee et al., 2002) and reduces motility of Haemonchus contortus and other parasites
in culture at concentrations ≥ 10-7 M (Lee et al., 2002; Zinser et al., 2002). It is highly active
against various species of parasitic nematodes, especially trichostrongylids, in vivo (Lee et al.,
2002). In this context, the almost complete lack of activity of 2-DOPH against C. elegans in
42 standard bioassays is puzzling. As this species expresses a binding site for the paraherquamide
class (Geary and Thompson, 2001), it is likely that the lack of activity is due to an unanticipated
barrier property of the C. elegans cuticle. Previous studies had suggested that the permeability
properties of several species in the Phylum Nematoda, including C. elegans, were generally
similar (Schaeffer et al., 1992), but this assumption has not been rigorously tested.
We used a cut worm assay (Lewis et al., 1980) to investigate the basis for the relative inactivity
of 2-DOPH. Cutting the worm preserved motor activity over a period of approximately 30 min
and removed the cuticle as a permeability barrier. The original studies showed that the potency
and time to paralysis of nicotinic agonists and the antagonist mecamylamine were markedly
enhanced in cut versus intact specimens of C. elegans (Lewis et al., 1980). We reproduced those
findings in the current work and extended the observations to include 2-DOPH and AAD 1470.
The new data support several conclusions. First, they confirm that the new anthelmintic 2-DOPH
is an nAChR antagonist in nematodes (Lee et al., 2002; Robertson et al., 2002; Zinser et al.,
2002; Qian et al., 2006), and that this activity extends to the free-living nematode C. elegans.
Although the preparation used for these experiments poses technical difficulties for precise
measurements of pharmacological responses, the activity of 2-DOPH as an nAChR antagonist
was clear. It appeared to antagonize the effects of bephenium better than the other agonists, with
least activity against levamisole. Paraherquamide anthelmintics also discriminate among nAChR
subtypes in Ascaris suum, the primary source of physiological and pharmacological data on
nematode nAChRs (Robertson et al., 2002; Levandoski et al., 2005; Qian et al., 2006; Robertson
and Martin, 2007). The pharmacology of nAChRs is clearly not identical in A. suum and C.
elegans (see Williamson et al., 2009). Whether these differences reflect phylogenetic distance
(Aguinaldo et al., 1997) or the adoption of a parasitic life style in A. suum remains unknown. The
therapeutic targets of the paraherquamides are primarily trichostrongylid nematodes of ruminants
(Lee et al., 2002; Little et al., 2010); these organisms are in the same clade as C. elegans
(Aguinaldo et al., 1997). Whether they more closely resemble C. elegans or A. suum in responses
to 2-DOPH has not been experimentally resolved. Our data show that 2-DOPH appears to act
preferentially on B-type nAChRs and is less potent against L-type nAChRs, as observed in A.
43 suum (Robertson et al., 2002; Qian et al., 2006), but these observations should be validated at the
single channel level in C. elegans.
The second conclusion from this work is that the new anthelmintics monepantel and derquantel
may not suffer from a negative interaction if used together in the treatment of nematodiases.
Similar to derquantel, monepantel is intended for use primarily for the control of trichostrongylid
nematodes of ruminants (Kaminsky et al., 2008; Hosking et al., 2010). The AAD class of
anthelmintics targets an unusual type of nAChR which has not been associated with responses to
other anthelmintic nAChR agonists (Kaminsky et al., 2008; Rufener et al., 2009). From the
current work, it seems that classical nAChR antagonists as well as agonists lack affinity for the
monepantel-sensitive receptor subtype. As resistance to currently available anthelmintics is a
serious global problem in small ruminants (Fleming et al., 2006); pressure to deploy these new
compounds may lead to their (unintended) use in concert. The data presented here suggest that
co-administration may not have negative consequences for parasite removal. However, this
observation obviously requires confirmation in target parasite species.
Finally, these results contribute to the perspective of recent efforts in anthelmintic discovery.
Many animal health companies used C. elegans as a primary screen for anthelmintic discovery
during the 1980s-1990s, but no new classes of anthelmintic were discovered in that system (the
prototype anthelmintics in every class were discovered in screens employing parasitized animals
or parasites in culture). In contrast, the prototypes of two new classes, paraherquamide and the
cyclic depsipeptide PF1022A, are essentially inactive in C. elegans bioassays (Conder et al.,
1995). Modification of PF1022A by the addition of morpholino substituents to achieve activity
in ruminants and more potent activity in monogastric animals generated the commercial product
emodepside (Scherkenbeck et al., 2002; Harder et al., 2003). Emodepside is also quite active
against C. elegans, in which it appears to act by prolonging the open time of a voltage-activated
potassium channel (Guest et al., 2007). The basis for this expansion in spectrum of activity has
not been resolved.
These data suggest that the permeability properties of the nematode cuticle vary across species,
or perhaps with lifestyle. Previous studies have shown that filariid cuticles are considerably less
44 of a permeability barrier than cuticles of gut parasites (see Ho et al., 1994), perhaps because
filariid parasites exist in host environments that are highly controlled in pH, osmolarity and other
chemical and physical properties. Similarly, parasites of the gastrointestinal tract face less
stringent environmental stresses than nematodes in the soil environment, including C. elegans
and free-living stages of parasitic species. Nematodes in soil environments would be expected to
exhibit more robust defences against xenobiotics than tissue stages of parasites. The range and
relative intensity of chemical insults from soil microbes will be much higher than in the intestinal
tract or internal tissue of a host; similarly, the range of environmental stresses is much higher
outside of than inside a host. It may not be surprising that cuticular permeability may differ to
address the demand for better protection, although measurements to test this hypothesis with
regard to C. elegans or larval parasites have not been reported (see Ho et al., 1994; Johnson et
al., 2004). The hypothesis is consistent with observations that L3 stages of trichostrongylid
nematodes are generally less sensitive to anthelmintics than adult stages (see Folz et al., 1987). It
is not possible at this point to empirically determine whether the emphasis on C. elegans as a
screening platform missed important actives in addition to the paraherquamides and cyclic
depsipeptides. However, a recent study on the accumulation of drug-like molecules by C.
elegans in culture revealed that 90% of these compounds failed to reach internal concentrations
of even 50% of the external level, suggesting that processes for restricting xenobiotic
accumulation may be much more prominent in this species than in target (adult stage) parasites
(Burns et al., 2010). Additional evidence to support this contention is provided by experiments
with the nAChR agonist amidantel and its major metabolite, de-acetylamidantel. The minimum
effective concentrations of amidantel and its de-acetylated metabolite in intact C. elegans were
350 and 180 μM, respectively, but these values decreased to 0.3 and 0.07 μM, respectively, in
cut worms (Tomlinson et al., 1985). However, both compounds are potent anthelmintics in
animal models of nematode infections (Xue et al., 2010), suggesting that their propensity for
accumulation is much greater in parasitic species compared with C. elegans.
It must also be recognized that the adoption of mechanism-based screening technologies for new
anthelmintics has not been successful in identifying new molecules on the market or in
development. With resistance to available drugs a continually expanding problem, new discovery
45 strategies are urgently required. Quantitative analyses of cuticular permeability in different
strains of C. elegans could be done to identify a mutant that expresses a cuticle more similar to
that of parasitic adults in this regard as one way forward. In any case, it is likely that historical
screening strategies which relied on initial activity against wild-type C. elegans failed to reveal
additional compounds with potentially useful anthelmintic properties.
Acknowledgments
This work was supported by an NSERC (Natural Sciences and Engineering Research Council of
Canada) summer scholarship to CV and by funds from NSERC, the Canada Research Chairs
program and the FQRNT (Fonds québécois de la recherche sur la nature et les technologies)
Centre for Host-Parasite Interactions (to TGG). ERL is supported by a Tomlinson Fellowship.
We thank Dr. Debra Woods of Pfizer Animal Health, Kalamazoo, MI, USA, for the gift of 2desoxoparaherquamide and Dr. Ronald Kaminsky of Novartis Animal Health, Basel,
Switzerland, for the gift of AAD 1470. We also thank the Caenorhabditis Genetics Center
(University of Minnesota, Minneapolis, MN, USA) for provision of the strain used.
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In the previous manuscript, I confirmed that bisected C. elegans worms preserve motor activity
and respond to pharmacological stimuli, producing responses controlled by the neuromuscular
system. These results validated the use of the cut-worm model to study neuropeptides in situ and
encouraged us to design a method to associate neuropeptides with their receptors in situ using C.
elegans cut-worm preparations. This initiative constitutes the initial scope of the subsequent
work and arises from the need to find novel strategies for neuropeptide – receptor pairing
(deorphanization) that overcome the limitations of heterologous receptor expression platforms
for this purpose. In the following chapter, I describe and evaluate the locomotion phenotypes
produced by physiologically-relevant FMRFamide-like peptides (FLPs) in C. elegans cut worms.
Subsequently, I use FLP phenotypes as a read-out in a low-throughput screen of receptors using
C. elegans strains with loss-of-function mutations in individual G protein-coupled receptor
(GPCR) genes. In a proof-of-concept experiment with a known FLP-GPCR pair, I questioned
whether it is possible to associate FLPs phenotypes with genotypes in these experimental
conditions and if the method discriminates among different C. elegans knockout strains and
consequently different receptors. Results of this validation as well as phenotype rescue
experiments in knockout strain prove the strategy, which allowed the identification of 9 new
FLP-GPCR associations. I further characterize one of them by receptor activation bioassays in
recombinant S. cerevisiae strains.
49 Chapter III. Manuscript II.
Novel approach for receptor deorphanization in the model nematode,
Caenorhabditis elegans
Elizabeth Ruiz-Lancheros1, Charles Viau1, Tita N. Walter1, Joseph A. Dent2, Gregor Jansen3,
David Y. Thomas3, Timothy G. Geary1,a
1
Institute of Parasitology, 2Department of Biology, 3Department of Biochemistry McGill University, Montreal - Quebec, Canada
a
Institute of Parasitology, 21111 Lakeshore Road, Ste-Anne-de-Bellevue – Quebec,
Canada, H9X 3V9
Manuscript in preparation
50 Abstract
FMRFamide-like peptides (FLPs) play critical roles in nematode nervous systems. G proteincoupled receptors (GPCRs) for which FLPs are ligands are likely to be good targets for
anthelmintic discovery. However, pairing FLPs with receptors in parasitic and free-living
nematodes has been problematic. Relatively few GPCRs have been matched (deorphanized) with
a cognate FLP(s) using reverse pharmacology. Here we describe a novel strategy to match FLPs
with their receptors directly in Caenorhabditis elegans bioassays. The method uses FLPs as baits
in situ to monitor specific loss of FLP-phenotypes in strains with loss-of-function mutations in
genes encoding individual candidate GPCRs. Phenotypes produced by profoundly active FLPs
like FLP-18, AF2, AF1, AF8 and 4 other FLPs were identified in cut worms (to bypass the
cuticular permeability barrier) and used as read-outs for a low-throughput receptor screening in
situ. Data from proof-of-concept studies using the deorphanized FLP-18R (NPR-5) show the
value of in situ strategy, which allowed the association of one or two GPCRs with each FLP. The
pharmacology of the AF2 receptor was further studied by expressing the identified GPCR in
modified strains of Saccharomyces cerevisiae for receptor activation bioassays. In situ method
identified FLP-GPCR pairs that were missed by other strategies and produced invaluable data for
ongoing efforts in anthelmintic discovery.
51 1.
Introduction
Anthelmintic resistance is a serious concern in human and veterinary medical settings, lending
urgency to the search for new drugs that escape existing resistance mechanisms to reinvigorate
the treatment of nematodiases (Nwaka and Hudson, 2006; Martin and Robertson, 2010).
Helminth neuropeptidergic systems are a promising source of drug targets, as they play critical
roles in worm biology and show conservation of key components in diverse free-living and
parasitic species (Mousley et al., 2004; McVeigh et al., 2012). Of particular interest are the
FMRFamide-like peptides (FLPs). FLPs are abundant in helminths and present in all target
invertebrates, including parasitic nematodes, flatworms and arthropods (McVeigh et al., 2005;
Walker et al., 2009; Marks and Maule, 2010). FLP sequences are broadly conserved among
species in these phyla, and few homologs have been identified in vertebrates (Maule et al., 2002;
Dockray, 2004; Walker et al., 2009). Some FLPs potently influence neuromuscular functions and
show cross-phylum activity, suggesting conservation of ligand-receptor recognition features
(Mousley et al., 2005). These characteristics make the FLP system an excellent drug target,
offering the possibility of low host toxicity and remarkably broad spectrum.
FLPs cannot be used as drugs for biopharmaceutical reasons. Instead, non-peptide ligands that
mimic or antagonize the effects of peptides are needed (Geary, 2010). FLP receptors are
intriguing targets for the discovery of non-peptide FLP ligands, as these receptors have no close
homologs in vertebrates and are expressed in therapeutically sensitive target tissues in
invertebrates (Geary, 2010). Most studies at the receptor level have been done in nematodes and
in flatworms. However, few receptors have been associated with their endogenous ligand(s)
(deorphanized) and only one parasitic nematode FLP-GPCR pair has been predicted (McVeigh et
al., 2006; Husson et al., 2007; Omar et al., 2007; Frooninckx et al., 2012; Atkinson et al., 2013).
Moreover, we have insights into the endogenous functions of only a few deorphanized receptors
(Janssen et al., 2010; Frooninckx et al., 2012).
It appears that almost all FLPs act through G-protein-coupled receptors (GPCRs) (Husson et al.,
2007). In Caenorhabditis elegans, 12 FLP-GPCRs (including some receptor isoforms) have been
deorphanized in heterologous expression systems through reverse pharmacology (Kubiak et al.,
52 2003a; Kubiak et al., 2003b; Lowery et al., 2003; Mertens et al., 2004; Mertens et al., 2005;
Mertens et al., 2006; Kubiak et al., 2007; Larsen et al., 2013). > 60 different orphan GPCRs have
been tested against more than 200 peptides in such systems (Lowery et al., 2003). However,
most profoundly bioactive FLPs remain unmatched to receptors, perhaps due to difficulties in
obtaining functional expression of invertebrate receptors in mammalian cells or Xenopus
oocytes, which makes problematic the measurement of their activation by peptides in standard
screening formats.
To overcome these limitations, we designed and validated a novel approach to match FLPs with
their cognate receptors in C. elegans bioassays; we focus our efforts on peptides that are
prominently involved in motor functions in parasitic species. Bioactive FLPs were used as baits
to search for FLP receptors in situ instead of in heterologous expression systems. Using cut
worms to bypass the cuticular permeability barrier, we identified characteristic FLP phenotypes.
We conducted a low-throughput screen in which we interrogated associations of FLP-phenotype
with specific receptor(s) in strains with loss-of-function mutations in candidate GPCR genes. As
a proof-of-concept, we confirmed the FLP18-6/NPR-5 ligand-receptor association. Furthermore,
the pharmacology of the AF2 receptor identified in the in situ screen was studied in receptor
activation assays in recombinant Saccharomyces cerevisiae strains. These results accelerate the
deorphanization process and a further study of these associations could provide a new set of
targets for anthelmintic discovery.
2.
Materials and Methods
2.1.
Materials
C. elegans wild-type (wt) N2 and mutant strains (Table S1) were kindly provided by the
Caenorhabditis Genetic Center (University of Minnesota, Minneapolis MN) and the National
Bioresource Project for the Nematode (Tokyo Women’s Medical University School of Medicine,
Tokyo). All strains were grown at 22°C in Petri dishes containing NGM (Nematode Growth
Medium) seeded with Escherichia coli strain OP50, and were synchronized by standard methods
(Stiernagle, 2006).
53 Peptides were custom-made by Sheldon Biotechnology Centre (McGill University). 1 mM stock
solutions were prepared in double-distilled water and kept at -20°C. FLP stocks were thawed just
before use to make 100 μM solutions in Artificial Perienteric Fluid (APF). APF is 67 mM NaCl,
67 mM NaCH3COOH, 3 mM KCl, 15.7 mM MgCl2, 3 mM CaCl2 and 5 mM Tris; glacial acetic
acid was added to reach pH 7.6 (Parri et al., 1991). FLP-APF solutions and dilutions were
prepared daily before experiments and maintained at 4°C. Peptidases inhibitors were added to
FLP-APF
solutions
to
limit
peptide
degradation.
phenylmethanesulphonylflouride
(Sigma)
alone
or
Amastatin,
in
phosphoramidon
combination
at
and
recommended
concentrations (Sajid et al., 1996; Sajid et al., 1997).
2.2.
Worm assay and FLP phenotype identification
Cut worm assays were performed using young adult stage C. elegans cultures, adapting
published protocols (Ruiz-Lancheros et al., 2011). Worms in 100 μl APF were bisected
approximately two-thirds from the anterior end using a blade thinly coated with silicon grease.
Animals with excessive tissue damage were rejected and only cleanly cut specimens were used.
Three wild type (wt) cut worms were transferred to a well with FLP-APF solution and monitored
for locomotion behaviour over approximately 30 min. Behaviours were scored until worms
failed to respond to nose tap, which was considered paralysis. 30 specimens per FLP-APF
solution were studied in three replicates using blind scoring with more than one observer to
avoid bias and ensure reproducibility. As controls, 10 specimens in APF were scored for
behaviours in parallel with specimens in FLP-APF solution. The most prominent and consistent
behaviour produced by a FLP-APF solution during the observation period, different from
behaviours in control worms, was selected as the relevant FLP phenotype. The time at which this
behaviour was observed was recorded as the onset time.
2.3.
Receptor selection
Peptide-GPCRs in the C. elegans database were searched using BLASTp and the Lymnaea
stagnalis lymnokinin-actived GPCR (AAD11810.1) as a query. GPCRs with e-value > 3e-07
were selected to perform different alignments to generate phylogenetic trees. Orphan GPCRs
from our search were aligned with deorphanized neuropeptide GPCRs to build phylogenetic
54 trees. Orphan GPCRs most closely related to deorphanized GPCRs and those that share the same
family tree in the TreeFam tool were selected as the candidate FLP-GPCRs (Li et al., 2006).
2.4.
Receptor screen in C. elegans
28 C. elegans mutant strains in which individual GPCR genes were knocked-out (KO) by
chemical mutagenesis were used for screening. Strains were maintained separately and studied in
groups on different days as recommended (Hart, 2006). Young adults were dissected as
described above. Three KO or wt cut worms were transferred to a well containing FLP-APF
solution and observed for the FLP-phenotype. Observations were done until the onset time of the
FLP-phenotype in wt worms or until paralysis. 10-12 specimens were studied per mutant strain
using as a control specimens from the same strain in APF solution without peptide. Fisher’s
exact test was used to detect significant differences between the number of wt worms and the
number of KO worms that showed the FLP-phenotype. Significance levels were set at p-Value 1Tail <0.05. The same procedure was used for all FLPs and mutant strains.
2.5.
Mutant rescue assay
To rescue a wt FLP phenotype in selected GPCR mutant strain, a transgene was microinjected
into mutant animals. The transgene was generated from wt gDNA by fusion PCR using a 3 Kb
region directly upstream of the initiation ATG, the full-length GPCR gene and its 3’-UTR as
described by (Boulin et al., 2006). Germline transformations using 10 ng/ul of DNA and 30 ng/ul
of the ttx3::gfp transformation marker were done following standard microinjection protocols
(Mello and Fire, 1995; Hobert et al., 1997; Evans, 2006). Transgenic animals were identified by
GFP fluorescence in the AIY interneurons at the pharynx (tt3x::gfp). At least two independent
transgenic cell lines were used for cut-worm experiments to assess the FLP phenotype in
transgenic animals, using as controls wt and mutant animals carrying the injection marker alone.
2.6.
Receptor activation assay in heterologous system
In situ deorphanized receptors were expressed in a collection of Saccharomyces cerevisiae
strains based on CY13193 (MATα PFUS1-HIS3 far1Δ1442 gpa1Δ1163 his3 leu2 lys2 trp1 ura3
sst2Δ2 ste14::trp1::LYS2 ste18γ6-3841 ste3Δ1156 tbt1-1), as described (Zhang et al., 2004; El55 Shehabi and Ribeiro, 2010; Larsen et al., 2013). Each strain contains an integrated modified copy
of the yeast Gα-subunit gene (GPA1) in which the last five amino acids of the protein were
replaced with those of various human Gα-subunits to express chimeras of the Gi-, G12-, Gq- G13-,
Gz and Gs-proteins or with those of 6 different C. elegans Gα-proteins. As reported previously
(Larsen et al., 2013)1 strains express HIS3 under control of the FUS1 promoter when an agonist
activates a heterologously expressed GPCR. HIS auxotrophy correction is detected by
monitoring yeast growth in Complete Minimal (CM) leucine and histidine dropout liquid
medium (CM/Leu-/His-), supplemented with 0.05 M MOPS, pH 6.8. Cell growth was measured
by Alamar Blue in a FlexStation plate fluorimeter reader (Molecular Devices) set at 560 nm
excitation/590 nm emission. Data were analyzed using GraphPad software (GraphPad, San
Diego, CA). Concentration–response curves were analyzed using nonlinear regression analysis
of each treatment run three times in triplicate and expressed as mean values ± 95% confidence
intervals (C.I.).
2.7.
Receptor expression and membrane localization in S. cerevisiae
To confirm the expression and cellular localization of recombinant C. elegans GPCRs in yeast,
we used activation of the High-Osmolarity Glycerol (HOG) pathway as a reporter. This pathway
is induced by a mitogen-activated protein (MAP) kinase cascade that leads to the synthesis of
glycerol to combat extracellular hyperosmotic stress. The yeast MAP kinase kinase kinase
(MAPKKK) ste11p is a key component of the cascade and is regulated and localized at the
plasma membrane by its interaction with Ste50 at its SAM (Sterile α Motif) domain (Posas et al.,
1998; Wu et al., 1999; Wu et al., 2006). To bypass the need for Ste50 in yeast cells (Ste50Δ
ssk2Δ ssk22Δ), and preserve ste11p functionality, we fused the recombinant GPCR to the
Ste11ΔSAM n-terminus and used the receptor as a Ste11 membrane-targeting signal.
Consequently, activation of the HOG pathway in high osmolarity media indicates that the GPCR
is in the plasma membrane.
1
See a full description of this manuscript and methods in Appendix. 56 We attempted to improve expression and cellular localization of C. elegans GPCRs by inserting
at the N-termini the signal sequence MSDAAPSLSNLFYDPTYNPG from the yeast GPCR Ste2
(YSS Ste2L), as was suggested for the human GPCR Frizzled receptors Fz1/Fz2 by Dirnberger
and Seuwen (2007). To build the chimera construct, we used the Gateway in vitro recombination
system (Invitrogen) to insert a C. elegans GPCR ORF (without stop codon) into a pDonor vector
and to transfer it to a Galactose (Gal) inducible yeast expression vector. The vector was derived
from the pGREG vectors reported by Jansen et al. (2005), and contains YSS Ste2L and
Ste11pΔSAM fragments flanking a Gateway cassette. Yeast strain YCW1476 (Ste50Δ ssk2Δ
ssk22Δ) was transformed using a quick lithium acetate method (Jansen et al., 2005) and selected
in CM Glucose - Uracil dropout medium (CM/Glu/Ura-). Induced positive clones in
CM/Gal/Ura- plates were incubated for 2 days at 30°C and tested for the ability to grow on
hyperosmotic media as suggested by Wu et al. (2006). The HOG assay was performed in
selective plates containing CM/Ura- supplemented with Gal and 1.25 M Sorbitol, and replicated
twice to clean background before scoring growth in high osmolarity media.
3.
Results
3.1.
FLP-phenotype identification
As previously reported (Ruiz-Lancheros et al., 2011), cut worms preserved motor activity for 20
- 30 min and became quickly paralyzed by anthelmintic cholinergic agonists or antagonists
(Lewis et al., 1980; Ruiz-Lancheros et al., 2011). This suggests that elimination of the cuticle as
a permeability barrier increases access of compounds to their target(s), and confirms that cut
worms respond to pharmacological stimuli and produce responses controlled by the
neuromuscular system. The cut preparation is therefore suitable for studying FLP effects.
Body movements of wt cut worms in solution differ from typical movements of intact specimens
and have not been reported in detail previously. We scored the behaviours of cut worms in APF
without peptide and in FLP-APF solutions. Peptide concentrations of 1 - 100 μM were tested and
peptidase inhibitors were added to FLP-APF solutions to limit peptide degradation. The most
consistent and fastest-onset phenotypes were observed at 100 μM peptide with or without
inhibitors. This concentration is high compared to the concentrations needed to observe peptide
57 effects in isolated A. suum muscle preparations (1 nM -10 μM, Maule et al., 1996). In A. suum
peptide microinjection studies, it was assumed that there is a 10-fold dilution in pseudocoelomic
fluid (Davis and Stretton, 2001). Since we were looking for rapid, profound and consistent
changes in behaviour after exposure to peptide and were unable to estimate the extent of FLP
diffusion to receptors in cut worms, we used 100 μM peptide concentrations in all in situ studies.
After scoring 30 wt specimens per FLP, we identified specific FLP-phenotypes that occurred
within an appropriate onset time (Table 1). For example, after 10 min in 100 μM AF2
(KHEYLRFa), wt cut worms straighten and develop an intense twitch, especially in the anterior
end. This phenotype was not observed in control cut worms, suggesting that these are specific
effects produced by AF2 (Video S1). The same phenotype was produced by 100 μM AF1
(KNEFIRFa), but more quickly. Three other FLPs, AF21 (AMRNALVRFa), AF22
(NGAPQPFVRFa) and FLP13-3 (APEASPFIRFa), produced quick paralysis, with worms
showing only rare sporadic movements of the head (Video S1). Of 14 FLPs tested, 6 showed no
behaviours that clearly differed from control cut worms.
Trivial
name1
PF1
FLP2-1
FLP3-1
Precursor
gene
flp-1
flp-2
flp-3
FLP sequence
FLP-phenotype
SDPNFLRFa
SPREPIRFa
SPLGTMRFa
ND3
ND
Curling
/disintegration
FLP4-2
AF8/PF3
flp-4
flp-6
ASPSFIRFa
KSAYMRFa
ND
Retracting
AF1
flp-8
KNEFIRFa
Twitching
FLP-9
AF21
flp-9
flp-11
KPSFVRFa
AMRNALVRFa
ND
Paralysis
FLP13-3
AF2
FLP18-6
AF22
SchistoFLP
PF4
flp-13
flp-14
flp-18
flp-11
unknown
similar in flp-1
APEASPFIRFa
KHEYLRFa
DVPGVLRFa
NGAPQPFVRFa
PDVDHVFLRFa
KPNFIRFa
Paralysis
Twitching
Curling
Paralysis
ND
ND
FLP-phenotype
description
Persistent body curls and looping/
Tissues and filaments protruding
from the anterior (cut) end
Quick retraction in body movement,
initially high bends following by
spasm
Body straight, few head movements
and intense body twitch
Body straight and worms do not
respond to tap
Persistent body curls and looping
onset
time2
--10
-5
5
-10
10
10
5
5
---
Table 1. FLP-phenotypes in C. elegans preparations. Bisected wt worms were exposed to FLP-AFP solutions.
Described phenotypes correspond to consistent behaviour observed in 30 specimens. FLP-phenotypes can be
visualised in Video S1. 1The name of some peptides reflects their initial isolation from other organisms.
2
Approximate phenotype onset time in minutes. 3No detected phenotype.
58 3.2.
Proof-of-concept
FLP18-6 (DVPGVLRFa) has been associated with three GPCRs: NPR-1 (C39E6.6), NPR-4
(C16D6.2), and two spliced forms of NPR-5 (Y58G8A.4) (Kubiak et al., 2003a; Rogers et al.,
2003; Kubiak et al., 2007; Cohen et al., 2009). Using the FLP18-6/NPR-5 pair, we investigated
whether this association would be apparent using phenotypes and a loss-of-function assay.
FLP18-6 produced pronounced body curls and loops in wt cut worms, which we denominated as
‘curling phenotype’ (Table 1, Video S1). npr-5(OK1583) mutant cut worms did not show the
curling phenotype in 100 μM FLP18-6, behaving the same as cut wt or npr-5(OK1583) worms
not exposed to FLP18-6 (Video S2). Cut worms from the same mutant strain tested with other
FLPs retained the relevant wt FLP-phenotypes (Fig. 1A). In addition, cut worms from other KO
strains, in which the unrelated genes eri-1 or rrf-3 are mutated, show the same curling phenotype
in FLP18-6 as the wt (Fig. 1B). This selectivity was also observed at the ligand level; FLP3-1
produced a phenotype similar to that produced by FLP18-6 in wt cut worms, but npr-5(OK1583)
worms retained the response to FLP3-1 (Fig. 1A). Additionally, transgenic strain expressing npr5 from its endogenous promoter in the npr-5(OK1583) background, recovered the curling
phenotype observed in wt cut-worms challenged with FLP18-6 (Fig 1C). We observed similar
results in two independent transgenic cell lines and no phenotype in mutant worms transformed
with injection marker alone. This confirms that the lack of wt curling phenotype in the KO strain
when challenged with FLP18-6 was associated with the loss of NPR-5 function and is not
mediated by other mutations in this strain. These findings validate the use of mutant strains with
loss-of-function mutations to screen candidate GPCRs using FLP phenotypes as the read-out.
59 Figure 1. FLP phenotypes in wt and mutant cut worms. A. C. elegans cut worms from wt and npr-5(OK1583)
mutant strains were exposed to peptides and scored for the corresponding phenotypes. B. FLP-18-6 curling
phenotype in wt and NPR-5 mutant strain (npr-5(OK1538)) compared with response of strains carrying mutations in
unrelated genes eri-1, Enhanced RNAi RNAse, (eri-1(mg366)), or rrf-3, RNA-dependent RNA-polymerase, (rrf3(pk1426)). C. FLP18-6 curling phenotype in wt, npr-5(OK1538) expressing transformation marker tt3x::gfp alone,
and transgenic cell lines L1and L2 expressing extrachromosomal arrays of npr5::npr5 in npr-5(OK1583)
background. Data are the percentage of worms that kept the phenotype (n = 10-12 specimens per peptide).
***P<0.0001, compared with wt or not significant when is not indicated.
3.3.
In situ screen
We chose the most likely FLP-GPCRs receptors to include in the in situ assay by bioinformatic
studies using BLASTp and TreeFam (Li et al., 2006). 21 orphan GPCRs and 7 previously
deorphanized GPCRs (Table S1) were included in the cut worm screen. For each, we used a
60 strain in which the receptor gene has been disrupted by random mutagenesis to an extent that
should create a null allele. It should be noted that some mutant strains used in this work were not
extensively out-crossed to isolate the GPCR mutation; the large number of strains employed in
the screen precluded this step for an initial screen. Nonetheless, the inclusion of multiple
peptides and strains, and the rescue of wt FLP phenotypes in selected mutant strains, controlled
for the presence of additional confounding mutations that could affect the interpretation of our
results.
Figure 2. AF2-receptor screening. 28 GPCR KO strains were screened for the AF2 (KHEYLRFa) twitching
phenotype. 10-12 cut worms per strain were tested with AF2. Results are expressed as percentage of worms that
kept the twitching phenotype observed in wt worms. GPCR KO strains carry mutations in genes receptors homologs
to Neuropeptide Receptors (npr), FMRFamide Peptide Receptors (frpr), Tachykinin-Like neuropeptide Receptors
(tkr), NeuroMedin U Receptors (nmur), Cholecystokinin-Like Receptors (ckr), Nematocin Receptor (ntr) and
Gonadotropin- Releasing Hormone receptor (gnrr). See Table S1 for further description of mutants. **P<0.01
compared with wt or not significant when it is not indicated.
Using 10-12 cut worms per KO strain in 100 μM AF2, we observed that worms from 26 KO
strains kept the wt twitching phenotype (Fig. 2). However, this phenotype was lost (85% of the
population) in the mutant frpr-18(ok2698), which carries a deletion in the T19F4.1 gene.
Comparison between wt and frpr-18 populations showed significant differences in the AF2
response (Table 2). These results indicate that FRPR-18 (FMRFamide Peptide Receptor-18) is
61 required for AF2 effects and is likely to be an endogenous receptor for AF2. FRPR-18 was
denominated FLP-2R because the only previously known ligands were FLP-2 peptides (Larsen et
al., 2013). Since we found another endogenous ligand for it, we return to the original
denomination as FRPR-18.
Furthermore, in two KO strains (npr-2 and frpr-18), only 9 and 17% of worms kept the AF1phenotype, respectively, suggesting that both NPR-2 and FRPR-18 are associated with AF1
responses (Fig. 3). AF8 (KSAYMRFa) produced retraction of body muscle in wt cut-worms
(Table 1). The only mutant that completely lost this phenotype was nmur-1(ok1387), which
carries a deletion in nmur-1 (Table 2 and Fig. S1A). Similarly, a single KO strain (npr-22) was
significantly different to wt and other strains in losing the response to FLP3-1 (SPLGTMRFa),
which induced a curling phenotype in wt cut worms (Table 2 and Fig. S1B). For other FLPs that
produced rapid paralysis in wt cut worms (AF21, AF22 and FLP13-3), we identified distinct KO
strains in which the respective response was lost (Table 2 and Fig. S2). The uncomplete lost of
phenotypes in KO strains correspond to variations into the population studied rather than
unspecific FLP interactions.
Figure 3. AF1-receptor screening. 28 GPCR KO strains were screened for the AF1 (KNEFIRFa) twitching
phenotype. 10-12 cut worms per strain were tested with AF1. Results are expressed as percentage of worms that
kept the twitching phenotype observed in wt worms. See Fig. 2 and Table S1 for description of mutants.
***P<0.0001, compared with wt or not significant when it is not indicated.
62 Name
AF2
AF1
in situ associated
receptor gene
frpr-18
FMRFamide peptide receptor
npr-2
neuropeptide receptor
frpr-18
Receptor
description
GPCR
FLP-2R
Orphan GPCR
GPCR
FLP-2R
Orphan GPCR
AF8/PF3
nmur-1
neuromedin U like receptor
FLP-3-1
npr-22
AF21
tkr-3
tachykinin-like neuropeptide
receptor
AF22
nmur-4
Orphan GPCR
FLP-13-3
npr-16
Orphan GPCR
gnrr-1
gonadotropin-releasing
hormone like receptor
Orphan GPCR
GPCR
FLP7R and FLP11R
Orphan GPCR
Mutant GPCR
%1
T19F4.1
(ok2698)
15
P-Value
1-Tail2
0.002
T05A1.1
(ok419)
T19F4.1
(ok2698)
9
0.000
17
0.000
5
0.000
18
0.030
25
0.001
4
0.000
17
0.000
50
0.012
C48C5.1
(ok1387)
Y59H11AL.1
(ok1598)
AC7.1
(ok381)
C30F12.6
(ok1381)
F56B6.5
(ok1541)
F54D7.3
(ok238)
Table 2. in situ deorphanized C. elegans GPCRs. FLP receptors were screened in C. elegans mutant strains using
as read-out FLP phenotypes. Listed are the receptors for which cut worms from the respective mutant strain lost the
phenotype observed in wt worms. Individual FLP-receptor screening graphs are shown in Fig. 2, 3, S1 and S2.
1
Percentage of worms that kept phenotype. 2 p-Value 1-Tail <0.05 compared with wt (Fischer’s exact test).
3.4.
Receptor activation in heterologous system
To study the pharmacology of associations found in situ, we used a yeast system in which the
recombinant GPCR is coupled to the endogenous yeast pheromone response (Dowell and Brown,
2002). NPR-5a expressed in this yeast system served as a positive control. NPR-5a was activated
in a concentration-dependent manner by FLP18-6 in yeast co-expressing a Gαq chimera, as was
observed in other expression systems (Kubiak et al., 2007; Cohen et al., 2009). We expressed the
receptor associated with AF2 in the in situ screen (FRPR-18) and challenged the recombinant
yeast with AF2 (100 μM). FRPR-18 was activated by AF2 when coupled with the Gαq chimera
in a concentration-dependent manner (Fig. 4). The EC50 value (14.7 μM, 95% C.I 11.7-18.4 μM)
was in the same range for NPR5a activation, and no activation was observed in strains
expressing other Gα chimeras or transformed with empty vector (mock) when challenged with
AF2. The activity of AF2 was receptor-specific since it was detected only in clones expressing
FRPR-18, which did not response to the FLPs listed on Table 2 (not shown). These results show
63 that the yeast system is suitable to study the pharmacology of FRPR-18, also indicate that the
AF2 receptor signals via Gαq and support the in situ deorphanization of FRPR-18 with AF2.
Two spliced forms of FRPR-18 (T19F4.1a and T19F4.1b) were deorphanized with FLP2-1 and
FLP2-2 (similar peptides ending in -EPIRFa) using reverse pharmacology (Mertens et al., 2005).
FLP-2 peptides also activated FRPR-18 in the yeast system in a concentration-dependent manner
when the receptor was coupled to the Gαq chimera (Larsen et al., 2013). However, in the in situ
test, FLP-2 peptides (100 μM) did not produce a detectable phenotype in cut wt worms or in
frpr-18(ok2689) worms. Furthermore, these peptides (100 μM) did not affect the AF2-twitching
phenotype in pre- or co- incubation experiments in cut worms (not shown).
Figure 4. Receptor activation in yeast by AF2. S. cerevisiae strains expressing chimeric Gαq and FRFR-18 or
empty vector (mock) were challenged with increasing concentrations of AF2. Cultures were grown in Complete
Minimal Leucine dropout media (CM/Leu-), and challenged with peptide in CM/Leu-/His- media. After 44 h at 30°C,
receptor activation was quantified using an Alamar Blue fluorescence assay. Data were normalized to the highest
value and are presented as the means and SEM of three experiments, each in triplicate.
All in situ deorphanized receptors were expressed in yeast following the same strategy; but none
was activated by the corresponding peptide associated in situ except FRPR-18. These despite the
use of yeast strains expressing 12 Gα chimeric proteins, including homologs of mammals Gαproteins and C. elegans Gα-proteins (not shown). As has been observed for other invertebrate
GPCRs, this lack of activity in the recombinant system can be influenced by poor receptor
expression and trafficking to the membrane in yeast (Levoye and Jockers, 2008).
64 3.5.
Monitoring C. elegans GPCR expression and plasma membrane localization in
yeast.
We used the HOG pathway to investigate the plasma membrane localization of recombinant
receptors in yeast. The lack of growth of GPCR-Ste11ΔSAM chimeras for receptors Fz2, NPR-5,
NPR-2 and FRPR-18 in hyperosmotic media (Fig. 5) suggested that the fusion proteins are either
not expressed or not translocated properly to the plasma membrane. In contrast, all YSS Ste2L
N-terminal tagged chimeras (Ste2L-GPCR-Ste11ΔSAM) were resistant to hyperosmotic stress
(Fig. 5), similar results were observed for the other in situ deorphanized receptors (not shown).
This indicates that the Ste2L chimera constructs were able to complement the wt ste11-ste50
function to activate the HOG pathway, suggesting that the C. elegans GPCRs were expressed
and localized properly. Similar results were observed for the human Fz2 receptor, which was
previously expressed in yeast using a longer Ste2 N-terminus peptide (Dirnberger and Seuwen,
2007). Results suggest that short peptides from endogenous yeast membrane proteins like YSS
Ste2L enhance plasma membrane expression of recombinant receptors, probably by facilitating
their proper co-translational insertion into the membrane.
Accordingly, Ste2L-GPCRs constructs were made for in situ deorphanized GPCRs and receptor
activation assays were performed. Ste2L-tagged NPR-5 and FRPR-18 produced similar EC50
values to untagged receptors when challenged with FLP-18 and AF2, respectively (not shown),
suggesting that the Ste2L fragment does not interfere with ligand binding. However, none of the
other Ste2L tagged GPCRs responded to the corresponding FLP even though their expression
and plasma membrane localization were confirmed (not shown).
65 Figure 5. Monitoring plasma membrane localization of heterologous GPCRs in yeast. Ste50Δ ssk2Δ ssk22Δ
yeast cells were transformed with Ste11ΔSAM fused with human GPCR Fz2 (A) or C. elegans GPCRs NPR-5 (B),
NPR-2 (C) and FRPR-18 (D), alone (no YSS) or carrying the yeast signal sequence YSS Ste2L at the receptor Nterminus. Clones were grown in CM glucose - uracil dropout media (Glu/Ura-) for 2 days at 30°C and assayed for
ability to restore the HOG pathway and grow in hyperosmotic media (Gal-Sorbitol/Ura-), using as controls empty
vector (mock) and untransformed strains.
4.
Discussion
We designed and validated a novel in situ strategy for matching nematode GPCRs with
endogenous ligands. 16 years after the C. elegans genome was reported, only 12 FLP- GPCRs
have been deorphanized despite the dedicated work of several laboratories in identifying FLPs
(McVeigh et al., 2005; Walker et al., 2009), and searching for their receptors using reverse
pharmacology (McVeigh et al., 2006; Husson et al., 2007; Frooninckx et al., 2012). In contrast,
the same strategy has deorphanized many GPCRs from humans and invertebrates, including
Drosophila (Lecca and Abbracchio, 2008).
66 The variety of neuropeptides in C. elegans (>200 peptides) and the number of orphan GPCRs (>
100 candidate GPCRs for non-odorant ligands) complicate the prioritization of GPCRs and
neuropeptides for more intensive study. Functional expression of nematode GPCRs in
heterologous systems can be problematic and it has been difficult to obtain stable transformed
mammalian cell lines for some (Mertens et al., 2005), necessitating special conditions to detect
functional receptors, such as lowering incubation temperature of transformants (Kubiak et al.,
2003b). Non-nematode recombinant systems may lack factors necessary for signalling of C.
elegans GPCRs (Levoye and Jockers, 2008). These issues have become a bottleneck in the
deorphanization and pharmacological characterization of neuropeptide GPCRs as anthelmintic
targets.
We adapted a different strategy to match ligands and receptors in situ. Neuropeptides were used
as baits for screening GPCRs. Our search focused on peptides with profound and potent activity
in A. suum physiological assays, assuming that this property would be generally conserved in C.
elegans (McVeigh et al., 2005; Mousley et al., 2005; McVeigh et al., 2006). Exposure to FLPs
produced no detectable effects in whole animals, probably because the size and hydrophilic
nature of FLPs limits diffusion through the C. elegans cuticle (Geary, 2010). Therefore, we
adopted the cut-worm model introduced by Lewis et al. (1980) and used reverse genetics to
interrogate association of specific GPCRs with FLP behavioural effects. Similar loss-of-function
studies have confirmed neuropeptide-receptor associations after their identification in a
heterologous system (Rogers et al., 2003). More recently, RNAi approaches have been proposed
to deorphanize receptors in flatworms (Zamanian et al., 2012), and in the plant parasite
Globodera pallida (Atkinson et al., 2013), using as a read out the effect on secondary
messengers and behavioural phenotypes, respectively. Here, we used loss-of-function studies for
peptide-receptor matching in C. elegans bioassays.
This strategy has several advantages. First, it gives more information about receptor – ligand
associations in situ. Second, it focuses on pairing physiologically relevant neuropeptides that are
prominently involved in motor functions in parasitic nematodes. This reduces the number of
GPCRs for subsequent pharmacological studies, focusing efforts to achieve functional
expression on a limited number of receptors. Finally, the strategy can be used to study other
67 components of the neuropeptidergic system, such as G-proteins associated with the receptors,
second messengers and other proteins in the signalling cascade.
Several technical issues (worm dissection, identification of a characteristic FLP phenotype and
use of multiple KO strains) limit the scalability of our strategy to screen the entire catalogue of
C. elegans GPCRs for matching ligands. However, it is well suited to screen for GPCRs that
mediate the effects of physiologically relevant FLPs among a small group of candidate receptors.
We included in this group GPCRs closely related to deorphanized GPCRs previously matched
with FLPs or NLPs, selected using a bioinformatic approach. It is also possible to select GPCRs
in silico using as a template deorphanized GPCRs and a MEST/MAST analysis (Multiple
Expectation Maximization for Motif Elicitation/Motif Alignment and Search Tool) to identify
characteristic neuropeptide-receptor motifs and GPCR candidates as proposed by Janssen and
collaborators (2010). Almost all GPCRs in our study are present in that MAST list (Janssen et
al., 2010; Frooninckx et al., 2012), supporting their inclusion.
The ligand-receptor pair FLP18-6/NPR-5 provided proof-of-concept; their association was
evident in loss-of-function assays in mutant strains and validated the in situ strategy. The loss of
the FLP18-6 phenotype in the npr-5 KO strain, the conservation of this phenotype in other KO
strains with loss-of-function mutations in unrelated genes, as well as the conservation of other wt
FLP phenotypes in the npr-5 KO, suggest that mutations in NPR-5 specifically disable the
FLP18-6 phenotype. This was confirmed by rescue of the FLP18-6 phenotype in transgenic npr5 mutant strains expressing NPR-5. Results of this series of experiments show that: mutation of
the endogenous GPCR disables peptide effects; the method discriminates among different KO
strains; and different FLPs and GPCR mutants can be used to screen receptors. Consequently, it
was possible to interrogate if a FLP that produces a reliable and distinct phenotype was
associated with a specific GPCR in a low-throughput screen. We found receptors for 7
previously unpaired FLPs and all showed the same stringent ligand-receptor selectivity detected
with the FLP18-6/NPR-5 pair.
We identified receptors for potent FLPs, including AF2, AF1 and AF8. More than 20 years after
their isolation and characterization (Cowden et al., 1989; Cowden and Stretton, 1993; Maule et
68 al., 1994), this is the first report of specific receptors associated with their action. AF2, AF1 and
AF8 play critical roles in neuromuscular physiology of some parasitic nematodes (Maule et al.,
1996; Davis and Stretton, 2001). Consequently, their receptors are intriguing drug targets. AF2,
AF1 and AF8 have also been isolated from C. elegans extracts (Marks et al., 1995; Marks et al.,
1998). They increased pharyngeal action potential frequency similar to serotonin (Rogers et al.,
2001) and stimulated pharyngeal pumping rate (Papaioannou et al., 2005); but their effects in C.
elegans locomotion have not been reported. In our cut-worm experiments, AF1 and AF2
produced paralysis and twitching, while AF8 produced characteristic body flapping. These
phenotypes could result from mixed inhibitory and excitatory effects in the C. elegans
neuromuscular system analogous to those observed in A. suum, a hypothesis that requires
experimental confirmation.
For each FLP we studied, the effect was lost in only one or two KO strains. In this regard, the in
situ matches identified two cases that reflect the complexity of the neuropeptidergic system.
First, some peptides were matched with more than one receptor; and second, one receptor was
associated with more than one peptide. In the first case, AF1 and FLP13-1 were each matched
with two receptors. This apparent ligand promiscuity was also observed using a traditional
approach for receptor deorphanization. For example, peptides encoded on flp-18 have been
reported to activate the receptors NPR-1, NPR-4 and NPR-5 (Rogers et al., 2003; Kubiak et al.,
2007; Cohen et al., 2009). Recognition of multiple GPCRs by a single peptide or peptide family
is commonly observed in mammalian systems as well (Geary, 2010), so this result is not without
precedent.
Furthermore, two receptors have been proposed to mediate the physiological effects of AF1 in A.
suum. The biphasic AF1 effects on body muscle strips were lost in denervated preparations, in
which only inhibitory effects were observed, suggesting that a muscle receptor mediates the
inhibitory effect and a neuronal receptor is responsible for excitatory responses (Maule et al.,
1996). Secondly, AF1 structure-activity studies shown that some amino acids are important for
the inhibitory but not the excitatory phase, suggesting involvement of more than one receptor
(Bowman et al., 1996; Thompson et al., 2003). Using C. elegans, we identified two GPCRs
associated with AF1, NPR-2 and FRPR-18.
69 The second case (one receptor for more than ligand) occurred for AF1 and AF2. frpr-18(ok2698)
worms lost the twitching phenotype produced by each peptide in wt worms. This kind of
multiple ligand association has previously been observed in other FLP-receptor pairs. For
example, NPR-1 is activated by FLP-21 peptides like GLGPRPLRFa (AF9) and by FLP-18
peptides like EMPGVLRFa. Both activated NPR-1 in a concentration-dependent manner and
differentially activated two isoforms, NPR-1-215F and NPR-1-215V (Rogers et al., 2003;
Kubiak et al., 2007). Receptor promiscuity can be explained by the conservation of specific
amino acids in the peptide sequences or by similarity in their tertiary structures in regions
essential for receptor recognition and activation, or by the existence of two independent ligand
binding sites on the same GPCR.
AF1 and AF2 are similar in sequence and produce similar physiological effects in C. elegans and
A. suum preparations, suggesting that they share a common receptor. However, studies in A.
suum suggest the opposite: Ala6-AF1 (KNEFIAFa) antagonized the effects of AF1, but not AF2,
on body wall muscle. Furthermore, the effects of AF1/AF2 chimeras were not equivalent to those
produced by the parent peptides (Bowman et al., 1996; Thompson et al., 2003) and AF1 did not
compete with [125I]AF2 in binding studies (Thompson et al., 2003). Finally, AF1 and AF2
produce different membrane responses in dorsal inhibitory A. suum motorneurons (Davis and
Stretton, 2001). Few data enable the confident extrapolation of ligand-receptor associations
across different nematode clades, and results from C. elegans may not be directly applicable to
A. suum and vice versa.
As a result of ligand-receptor promiscuity, we associated unpaired FLPs with previously
deorphanized receptors. For example, we matched FLP3-1 with NPR-22, which had been
associated with peptides encoded on flp-7 and flp-11 (Mertens et al., 2006). We also associated
AF2 and AF1 with FRPR-18, previously associated with two FLP-2 peptides (Mertens et al.,
2005; Larsen et al., 2013). The FLP-2 peptides activate FRPR-18 when expressed in mammalian
cells with EC50 values < 10 nM; EC50 values for these peptides were more than 10 times higher
when the receptor was expressed in yeast (Larsen et al., 2013). Here we observe that AF2
activates FRPR-18 in yeast co-expressing the Gαq chimera in a concentration-dependent manner
with an EC50 in the μM range, suggesting that this peptide is also a cognate ligand of FRPR-18.
70 EC50 values obtained in the yeast and mammalian cells are not comparable, due in part to
limitations in peptide permeability imposed by the yeast cell wall, which also differ between
peptides.
AF2 activated C. elegans FRPR-18 in yeast only in the presence of chimeric Gαq; no response
was observed with other chimeric Gα-proteins. AF2 acts through G-protein activation in A. suum
body wall muscle (Kubiak et al., 2003c), but no responsive GPCR or Gα-protein has been
reported in A. suum. AF2 strongly increased cAMP levels in A. suum specimens (Reinitz et al.,
2000) and muscle preparations (Reinitz et al., 2000; Thompson et al., 2003), suggesting that this
increase may be a secondary effect of AF2 receptor activation through Gαs. However, changes in
cAMP produced by AF2 are uncoupled to AF2 biphasic tension responses, and the increase in
cAMP is not necessarily the cause of AF2 excitatory effects in A. suum (Thompson et al., 2003).
The mechanism by which AF2 changes cAMP levels is not clear. AF2 also increases inward
Ca2+ currents in the bag region of A. suum muscle (Verma et al., 2007). It is possible that the Gproteins released upon GPCR activation by AF2 modulate voltage-gated Ca2+ channels in the
postsynaptic membrane and so potentiate muscle contraction in A. suum, (Catterall, 2000) but
this remains to be proven.
Based on results obtained in the yeast system, AF2 and FLP-2 peptides are ligands for the C.
elegans GPCR, FRPR-18. Nonetheless, in our cut-worm assay, FLP-2 peptides produced no
characteristic phenotype in wt worms. In contrast, AF2 was strongly associated with this receptor
in the heterologous system and in the in situ assay. On the other hand, AF1 was associated with
FRPR-18 and NPR-2 in the in situ screen (Table 2), but no activation of these receptors
expressed in yeast individually or together was observed. This result is difficult to understand in
light of the fact that we confirmed the heterologous expression and membrane localization of
each receptor using as a reporter the yeast HOG pathway. It is unknown if AF1 can indirectly
activate one of the receptors or if receptors must form heterodimers in vivo. It is also possible
that the heterologous system lacks specific C. elegans GPCR-interacting proteins for these
receptors be functionally active or that receptors do not activate the chimeric heterotrimeric Gproteins used in the yeast assay. These observations also apply to other in situ deorphanized
GPCRs for which activation by the corresponding peptide was not observed in yeast even when
71 expression in the plasma membrane was documented. Our results call into question the reliability
of heterologous receptor expression platforms for matching and support the use of in situ
deorphanization approaches despite their limitations for pharmacological studies.
Results from this study show that it is possible to associate ligands with receptors in situ and
identify strong FLP-GPCR pairs. Considering that neuropeptide GPCRs have been accepted as
legitimate targets for anthelmintic discovery, and indeed were explored as such in at least one
industrial operation (Woods et al., 2010), the new associations can enhance the search of
possible non-peptide ligands for FLP-GPCRs as a source of new anthelmintics with broad
spectrum of action and a reduced rate of resistance development based on receptor-level
mutations (Geary et al., 2012; Geary and Ubalijoro, 2012). Screening with matched FLP-GPCR
pairs will be enhanced by the addition of newly deorphanized GPCRs, especially those which
mediate physiologically profound neuromuscular responses. Thus, results from this work can be
rapidly translated into new drug discovery efforts.
Acknowledgments
This work was supported by an NSERC summer scholarship to C.V. and by funds from NSERC,
the Canada Research Chairs program, the FQRNT Centre for Host-Parasite Interactions, the Bill
and Melinda Gates Foundation, CIHR and the Grand Challenges Canada program (to T.G.G.).
E.R.L. is supported by a Tomlinson Fellowship. We thank J. Broach (Cadus, Inc., Princeton, NJ)
and Pfizer Animal Health (now Zoetis, Inc., Kalamazoo, MI) for providing S. cerevisiae strains.
We also thank the Caenorhabditis Genetics Center and the National Bioresource Project for the
Nematode for provision of strains.
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Gene name
C10C6.2
Receptor gene
denomination
npr-2
Neuropeptide
receptor family
npr-3
C16D6.2
npr-4
Y58G8A.4
npr-5
F41E7.3
npr-6
GPCR
FLP-15R
GPCR
FLP-4R and FLP-18R
GPCR
FLP-18R
Orphan GPCR
F35G8.1
npr-7
Orphan GPCR
RB761
C56G3.1
npr-8
Orphan GPCR
RB1321
ZK455.3
npr-9
Orphan GPCR
IC683
C53C7.1
npr-10
RB1325
C25G6.5
npr-11
T22D1.12
npr-12
GPCR
FLP-3R
GPCR
FLPsR
Orphan GPCR
ZC412.1
npr-13
Orphan GPCR
T27D1.3
npr-15
Orphan GPCR
RB1429
F56B6.5
npr-16
Orphan GPCR
RB1365
Y59H11AL.1
npr-22
RB1405
T19F4.1
frpr-18
FMRFamide
Peptide Receptor
family
frpr-10
GPCR
FLP-7R and FLP-11R
GPCR
FLP-2R
Orphan GPCR
RB1349
Orphan GPCR
VC2171
C49A9.7
tkr-1
tachykinin-like
neuropeptide
receptor
tkr-2
Orphan GPCR
RB1423
AC7.1
tkr-3
Orphan GPCR
VC270
T05A1.1
F57H12.4
C38C10.1
Ligand
associated
Orphan GPCR
Knockout
strain
XA3702
RB1393
RB799
RB2040
mutant
npr-2(ok419)
IV
npr-3(tm1583)
X
npr-4(tm1782)
X
npr-5(ok1583)
V.
npr-6(tm1497)
X
npr-7(ok527)
X
npr-8(ok1439)
X
npr-9(tm1652)
X
npr-10(ok1442)
X
npr-11(ok594)
X
npr-12(tm1498)
IV
npr-13(tm1504)
V
npr-15(ok1626)
III
nrp-16(ok1541)
X
Receptor selection
parameters
npr-1 paralog
Deorphanized FLP-GPCR
Deorphanized FLP-GPCR
Deorphanized FLP-GPCR
Phylogenetically related to
known FLP receptors
npr-3 paralog
Phylogenetically related to
known FLP receptors
npr-16 paralog in
TreeFam
Deorphanized FLP-GPCR
Deorphanized FLP-GPCR
npr-4 paralog in TreeFam
npr-5 paralog
Phylogenetically related to
NLP-12R
npr-9 paralog in TreeFam
npr-22(ok1598)
IV
frpr-18(ok2698)
V
Deorphanized FLP-GPCR
frpr-10(ok1504)
IV
tkr-1(ok2886)
III
FLP-2R paralog in
TreeFam
Phylogenetically related to
npr-22 in TreeFam
tkr-2(ok1620)
I
tkr-3(ok381)
IV
Phylogenetically related to
npr-22 in TreeFam
npr-22 paralog in
TreeFam
Deorphanized FLP-GPCR
77 T23B3.4
Y39A3B.5
C48C5.1
ckr-1
cholecystokininlike receptors
ckr-2
K10B4.4
nmur-1
Neuromedin U
Receptor homolog
nmur-2
C30F12.6
nmur-4
F54D7.3
gnrr-1
GonadotropinReleasing hormone
receptor homolog
ntr-1
Nematocin
Receptor orthologous to the
human gene
AVPR2
ntr-2
T07D10.2
F14F4.1
Orphan GPCR
GPCR
NLP-12R
Orphan GPCR
RB1923
RB1288
GPCR
NLP-44R
Orphan GPCR
RB2526
Orphan GPCR
RB509
Orphan GPCR
RB2105
Orphan GPCR
RB1284
ckr-1(ok2502)
I
NLP-12R paralog
ckr-2(tm3082)
III
nmur-1(ok1387)
X
Deorphan NLP-GPCR
nmur-2(ok3502)
II
nmur-4(ok1381)
I
gnrr-1(ok238)
I
NLP44R and NLP-12R
paralogs in TreeFam
Deorphan NLP-GPCR
NLP44R and NLP12R
paralogs in TreeFam
Phylogenetically related to
known NLP/FLP
receptors
ntr-1(ok2780)
I
Phylogenetically related to
NLP/FLP receptors
ntr-2(tm2243)
X
Phylogenetically related to
NLP/FLP receptors
Table S1. Selected receptors for screening. Receptors were selected after phylogenetic analyses of orphan and
deorphanized receptors for small molecules in C. elegans as well as analysis of tree families in TreeFam
(http://www.treefam.org).
78 Figure S1. AF8 and FLP3-1 receptor screening. 28 mutant strains carrying KO of different GPCRs were screened
using as a read-out the FLP phenotype. 10-12 cut worms per strain were tested with AF8 (A) and FLP3-1 (B).
Results are expressed as percentage of worms that kept the phenotype observed in wt cut worms. See Table S1 for
description of each mutant. ***P<0.0001, P*<0.05 compared with wt or not significant when it is not indicated.
79 Figure S2. AF22, FLP13-3 and AF21 receptor screening. 28 mutant strains carrying KO of different GPCRs were
screened using as a read-out the paralysis phenotype produced by different FLPs. 10-12 cut worms per strain were
tested with AF22 (A), FLP13-3 (B) and AF21 (C). Results are expressed as percentage of worms that kept the
phenotype observed in wt cut worms. See Table S1 for description of each mutant. ***P<0.0001, P**<0.01
compared with wt or not significant when it is not indicated.
80 SI Videos
https://www.videosprout.com/video?id=61d00902-2160-4fad-b3a9-248d163e1637
Video S1. FLP phenotypes in wt cut worms. Nine video segments show (A) wt behavior in APF
without peptide; (B, C) twitching phenotypes produced by AF2 and AF1, respectively; (D) the
AF8 retracting phenotype; (E-G) paralysis produced by AF21, AF22 and FLP13-3, respectively;
(H-I) the curling phenotype produced by FLP18-6 and FLP3-1, respectively. All peptides were
tested at 100 µM in APF; videos were taken in the same conditions and presented in 5 fps.
https://www.videosprout.com/video?id=53f5dd24-5c73-4735-b8e6-276a93199951
Video S2. wt and FLP-18R (Y58G8A.4) KO cut worm phenotypes. Four video segments show
(A) wt behavior in APF without peptide; (B) wt curling phenotype in FLP-8-6 100μM; (C)
response of FLP-18R OK cut worms to the same peptide; and (D) conservation of twitching
phenotype in the KO in AF2 100 µM. Videos were taken in the same conditions and presented in
5 fps.
81 Connecting Statement III
In manuscript II, I described the in situ strategy that allowed identification of new FLP-GPCR
associations that were missed in heterologous receptor expression platforms. I also identified
multiple associations that illustrate the complexity of the nematode neuropeptidergic system.
That complexity and the possible functional redundancy of peptides and receptors is relevant in
the search for or design of drugs that target deorphanized FLP-GPCRs. Non-peptide ligands for
neuropeptide GPCRs should be promiscuous, acting on multiple receptors and influencing
different FLP pathways, to maximize the impact on parasite biology. Importantly, a promiscuous
non-peptide drug will escape receptor-mediated resistance mechanisms and should also have a
very broad spectrum of action, considering the conservation of FLPs and their receptors across
helminth Phyla. To accelerate the rational selection for further development of promiscuous nonpeptide ligands that act as agonists or antagonists of neuropeptide GPCRs, a priority is to
investigate receptor interactions with endogenous ligand(s). This includes developing a better
understanding of the mechanisms by which one peptide ligand can activate different receptors
and one receptor is able to recognize different peptide ligands. In the following manuscript, I use
experience gained in in situ bioassays and heterologous expression systems (in vitro bioassay) to
characterize ligand-receptor interactions by structure-activity relationship (SAR) studies in situ
and in vitro. The well characterized FLP-18/NPR-4 and FLP-18/NPR-5 ligand/receptor pairs
serve as test models since these receptors are intriguing targets for anthelmintic discovery.
82 Chapter IV. Manuscript III.
Structure-activity relationships of Caenorhabditis elegans FLP-18 on the
G protein-coupled receptors NPR-4 and NPR-5
Elizabeth Ruiz-Lancheros, Timothy G. Geary
Institute of Parasitology, McGill University,
21111 Lakeshore Road, Ste-Anne-de-Bellevue QC Canada H9X 3V9
Manuscript in preparation
83 Abstract
The neuropeptidergic system of nematodes plays critical roles in modulating neuronal networks
directly or indirectly and consequently influences all nematode behaviors. Pharmacological
characterization of ligand-receptor interactions is a crucial step to understanding the multiple
ligand-receptor physiological associations observed in the nematode neuropeptidergic system
and for identifying targets for the discovery of novel anthelmintics. To gain insight into how
FLP-18 peptides encoded on the flp-18 precursor interact with the Caenorhabditis elegans G
protein-coupled receptors (GPCRs) NPR-4 and NPR-5, we used heterologous expression of
receptors in yeast and an in situ C. elegans bioassay for structure-activity relationship (SAR)
studies. Using truncated analogs of FLP18-6 (DVPGVLRFa) and an alanine scan series
(systematic replacement of each amino acid by alanine); we observed that PGVLRFa is the
minimal fragment needed for NPR-5a activation in vitro and in situ, while shorter amidated
peptides can activate NPR-4 in vitro. Alanine substitutions at the FLP18-6 C-terminus showed
the essential role of the –VLRFa motif in receptor interactions and suggested that residues L6,
F8 and the C-terminal amide determine activity at both receptors in vitro and NPR-5 in situ.
Other alanine substitutions showed that P3 modification affects potency at both receptors and
that V5 is differentially recognized by the receptors. Additionally, we found that alanine
substitution for D1 results in a more potent agonist of both receptors. These data illuminate the
slight differences in how NPR-4 and NPR-5 interact with FLP-18 peptides at the molecular level
and can support the search for non-peptide ligands as candidate anthelmintics.
84 1.
Introduction
For several reasons helminth neuropeptide G protein-coupled receptors (GPCRs) are legitimate
targets for anthelmintic discovery (see Greenwood et al., 2005; Geary, 2010; McVeigh et al.,
2012). First, neuropeptides play roles in every recognized behavior in worms, including
locomotion, feeding, reproduction, gustatory associative learning and memory (Li and Kim,
2010; Taghert and Nitabach, 2012). Neuropeptides in the FMRFamide-like peptide (FLP) family
potently influence the neuromuscular system, a known target for anthelmintics (Mousley et al.,
2010). Second, FLPs have few homologs in vertebrates, are conserved in many target
invertebrates and have shown cross-phylum activity, suggesting conservation of FLP functions,
receptors and ligand-receptor recognition features among arthropods and helminths (McVeigh et
al., 2005; Mousley et al., 2005; Marks and Maule, 2010). Third, FLP receptors are mainly
GPCRs, highly druggable targets amenable to high throughput screening platforms (Geary and
Ubalijoro, 2012; McVeigh et al., 2012).
Pairing neuropeptides and receptors (deorphanization), understanding biological functions of
receptors and the molecular interactions of neuropeptide ligands with their receptors are top
priorities to facilitate the search for or design of anthelmintics that target neuropeptide GPCRs.
Several deorphanization programs using reverse pharmacology and in situ studies in
Caenorhabditis elegans have shown that peptides encoded on different genes can activate the
same receptor, and that more than one distinct receptor can recognize the same peptide (RuizLancheros et al., unpublished observations2; Frooninckx et al., 2012). Moreover, physiological
studies in helminths and arthropods have suggested functional redundancy of peptides and
receptors (Mousley et al., 2004; Mousley et al., 2005), which illuminates the complexity of the
neuropeptidergic signalling system (Kubiak et al., 2007; Li and Kim, 2008). To better understand
this complexity and discover drugs that exploit receptor promiscuity, it is important to
characterize the pharmacological bases of ligand-receptor interactions and the mechanisms that
govern promiscuous interactions.
2
See a full description of this manuscript and methods in Chapter III 85 Structure-activity relationship (SAR) analysis is one approach to identify the amino acid residues
that are essential for biological activity by governing the interaction of peptides with endogenous
receptors. We investigated the SAR of DVPGVLRFa (FLP18-6) on its endogenous GPCRs
NPR-4 and NPR-5. –PGVLRFa peptides, encoded on the C. elegans flp-18 precursor gene,
potently induce profound locomotor responses in intact Ascaris suum and C. elegans
preparations (Ruiz-Lancheros et al., unpublished observations; Davis and Stretton, 2001). They
also induce excitatory effects on A. suum somatic and ovijector muscle tissues without inducing
marked changes on membrane potential and input resistance in DE2 and DI motor neurons
(Fellowes et al., 2000; Davis and Stretton, 2001; McVeigh et al., 2006). Silencing of the flp-18
ortholog in the plant parasitic nematode Globodera pallida induces an aberrant behavioral
phenotype (Kimber et al., 2007), while a C. elegans loss-of-function flp-18 mutant, flp-18(lf), has
defects in chemosensation, foraging, dauer formation, and fat accumulation and exhibits reduced
aerobic metabolism (Cohen et al., 2009). flp-18 overexpression induces an uncoordinated
phenotype and increases body curvature (Rogers et al., 2003). Reverse pharmacological studies
have demonstrated that FLP-18 peptides are potent ligands of NPR-4, two spliced forms of NPR5, and also NPR-1 (Rogers et al., 2003; Kubiak et al., 2007; Cohen et al., 2009).
The expression of flp-18 in the AIY and RIG interneurons has been associated with the
coordinated regulation of odor responses, foraging strategy and fat metabolism through NPR-4
and NPR-5 receptors. The npr-4(lf) mutant phenocopies the chemosensation, foraging, and fat
accumulation defects of the flp-18(lf) mutant, while the npr-5(lf) mutant has the same dauer
formation and fat accumulation defects of the flp-18(lf) mutant. These observations agree with
the expression of NPR-4 in intestine and AVA and RIV neurons, and with the expression of
NPR-5 in ciliated sensory neurons (Cohen et al., 2009). Recent findings have also suggested that
FLP-18 peptides together with FLP-1 peptides function in a homeostatic manner to modulate
output of the locomotor circuit. flp-18 overexpression in cholinergic motor neurons modulates in
part the excitation - inhibition imbalance in the locomotor circuit produced by gain-of-function
(gf) mutation in the nicotinic acetylcholine receptor ACR-2, acr-2(gf). FLP-18 peptides are
thought to act directly on the muscle to coordinate the activity state of the locomotion circuit;
86 this coordination involves NPR-5 and NPR-1, FLP-1 peptides interaction with unknown
receptors and other neuropeptides (Stawicki et al., 2013).
The promiscuous nature of FLP-18 peptides, the functionality of C. elegans FLP-18 receptors in
different biological roles and the conservation of these peptides and receptors among parasitic
nematodes (McCoy et al., 2014), make the FLP-18 receptors good drug target. They also provide
an excellent model to investigate ligand-receptor interactions, facilitating the design of
promiscuous non-peptide ligands as potential antiparasitic drug candidates. Here we test the
effects of FLP18-6 analogs, including truncated and alanine-scan series, in NPR-4 and NPR-5a
receptors in vitro, using Saccharomyces cerevisiae as an expression system, and in situ in C.
elegans bioassays.
2.
Materials and Methods
2.1.
Materials
DVPGVLRFa (FLP18-6), C-terminally and N-terminally truncated analogs, and an alanine scan
series were custom-made by Sheldon Biotechnology Centre (McGill University). Verification of
authenticity and purity was obtained via mass spectrometry. Stock solutions (1 mM) were
prepared in double-distilled water and stored at -20°C. For in situ assays, 100 μM peptide
solutions were prepared in Artificial Perienteric Fluid (APF) (67 mM NaCl, 67 mM
NaCH3COOH, 3 mM KCl, 15.7 mM MgCl2, 3 mM CaCl2 and 5 mM Tris, pH 7.6; (Parri et al.,
1991).
C. elegans wild-type (wt) N2 and npr-5(ok1583) and npr-4(tm1782) mutant strains were
provided by the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis MN) and
the National Bioresource Project for the Nematode (Tokyo Women’s Medical University School
of Medicine, Tokyo). Strains were grown at 22°C in Petri dishes containing NGM (Nematode
Growth Medium) seeded with Escherichia coli strain OP50. Young adult C. elegans cultures
were used for in situ assays.
87 2.2.
In vitro heterologous expression in S. cerevisiae
The yeast heterologous expression system described previously by Wang et al. (2006) was used
for in vitro SAR studies. Briefly, npr-4 and npr-5a ORFs were sub-cloned into the Cp4258
LEU2 selectable marker yeast expression vector. Selected clones were used to transform yeast
strains CY13397 and CY13393, respectively, using standard lithium acetate method as described
by (Jansen et al., 2005). These strains are based on parental yeast MATα PFUS1-HIS3 far1Δ1442
gpa1Δ1163 his3 leu2 lys2 trp1 ura3 sst2Δ2 ste14::trp1::LYS2 ste18γ6-3841 ste3Δ1156 tbt1-1,
and express Gαq and Gαi/o chimera proteins, respectively. In transformed strains, HIS3
expression under control of the FUS1 promoter occurs when the recombinant receptor is
activated, and HIS auxotrophy correction induces cell growth that is agonist concentrationdependent (Dowell and Brown, 2002; Szekeres, 2002). As described by Larsen et al. (2013)
transformants were challenged with increasing concentrations of each peptide in Complete
Minimal Leucine and Histidine dropout liquid medium (CM/Leu-/His-) supplemented with 0.05
M MOPS - pH 6.8, and cell growth was measured by Alamar Blue in a Synergy H4 Hybrid
Multi-Mode Microplate Reader (BioTek). Each treatment was run three times in triplicate using
the parent peptide FLP18-6 as control. Concentration–response curves were analyzed by
nonlinear regression analysis using GraphPad software (GraphPad, San Diego, CA).
2.3.
In situ C. elegans bioassay
Cut worm assays were performed as described (Ruiz-Lancheros et al., 2011). Briefly, worms in
100 μl APF were bisected approximately two-thirds from the anterior end using a razor blade.
Clean cut-worms were transferred to a well with 100 μl peptide solution to monitor the FLP-18
curling phenotype previously scored in wt cut-worms exposed to FLP18-6 (Ruiz-Lancheros et
al., unpublished observations). 10 specimens per peptide solution were studied and monitored for
5 min (curling phenotype onset-time). As controls, 10 specimens in APF were scored for
behaviours in parallel with specimens in peptide solution. Fisher’s exact test was used to detect
significant differences between the number of cut worms that showed the curling phenotype in
the parent peptide and the number of cut worms with the same phenotype exposed to FLP18-6
analogs. Significance levels were set at p-Value 1-Tail <0.05.
88 3.
Results
3.1.
NPR-4 and NPR-5a heterologous expression in S. cerevisiae
NPR5 and NPR-4 GPCRs were previously associated with FLP-18 peptides by reverse
pharmacology using mammalian cells and Xenopus oocytes as heterologous expression systems.
In both systems, NPR-5 isoforms a and b were activated equipotently by FLP18-6 and coupled
with the Gαq signaling cascade (Kubiak et al., 2007; Cohen et al., 2009). In contrast, NPR-4
required co-expression of a G-protein-regulated inward-rectifier K+ (GIRK) channel to respond
to FLP-18 peptides in Xenopus oocytes, suggesting that NPR-4 couples with a Gαi/o-protein
(Cohen et al., 2009). We expressed NPR-4 and NPR-5a in S. cerevisiae strains that co-express
chimeras of the corresponding Gα-proteins and challenged them with increasing concentrations
of FLP18-6. Both receptors respond to FLP18-6 in a concentration-dependent manner with EC50
values of 0.48 and 1.93 μM and 95% C.I (confidence intervals) of 0.44 - 0.52 and 1.80 - 2.08
μM, respectively (Fig. 1). No activation of NPR-5a was observed in clones transformed with
empty vector or in strains expressing other Gα chimera proteins (not shown). NPR-4 coupled
with chimeras of Gαi/o and Gαz proteins without marked differences in EC50 values (not shown).
Figure 1. Concentration-response curves and EC50 values of FLP18-6 tested on NPR-4 and NPR-5a receptors
expressed in yeast. A. NPR-4 activation by FLP18-6 in yeast co-expressing Gαi/o chimera protein. B. NPR-5a
activation by FLP18-6 in yeast co-expressing Gαq chimera protein. Growth induced by different concentrations of
FLP18-6 in CM/Leu-/His- media was measured in Alamar blue fluorescence assay. Data were normalized to the
highest value and are presented as mean and SEM of three experiments, each in triplicate.
89 3.2.
3.2.1.
FLP-18 analog effects in the in vitro bioassay
Truncated analogs
To identify the minimal core sequence required to activate NPR-4 and NPR-5a, we screened Nterminally and C-terminally truncated series of FLP18-6 in S. cerevisiae. We observed that the
amidated peptide sequences PGVLRFa and GVLRFa were agonists of NPR-4; both peptides
were less potent than FLP18-6, but elicited the same maximum response (Emax) as the parent
peptide (Fig. 2A). The amidated tetrapeptide sequence VLRFa only activated the receptor at
concentrations > 5 µM and the Emax was only 40% of that produced by longer peptides. The only
N-terminally truncated analog that was an agonist of NPR-5a was PGVLRFa, which was less
potent than FLP18-6 but induced the same maximum response (Fig. 2B). No other analog, at
concentrations of 100 µM, activated NPR-5a, which suggests a key role for the proline in
position 3 (P3) for NPR-5 agonism. For both receptors, amide truncation at the C-terminus,
DPGVLRF-OH, eliminated activity (Fig. 2A, B).
Figure 2. Concentration-response curves of N-terminally and C-terminally truncated FLP18-6 analogs on
receptors expressed in yeast. NPR-4 (A) and NPR-5a (B) were challenged with increasing concentrations of
FLP18-6, DVPGVLRFa (■), PGVLRFa (●), GVLRFa (), VLRFa (d) or DVPGVLRF-OH (c). Growth in
CM/Leu-/His- media was measured by Alamar blue fluorescence. Data were normalized to the highest value and are
presented as mean and SEM of three experiments, each in triplicate.
90 3.2.2.
Alanine substitutions
To study the role of each residue in receptor activation, we tested an alanine scan series of
FLP18-6 in which each residue was systematically replaced by alanine, D1V2P3G4V5L6R7F8a.
Alanine substitutions in the first 4 residues (N-terminal alanine modifications) of FLP18-6
induced shifts in the concentration-response curves for NPR-4 and NPR-5a (Fig. 3A, B). The
concentration-response curve of the Ala1 modification, AVPGVLRFa, was left-shifted compared
to the parent peptide in both receptors, suggesting that Ala1 is a more potent agonist of FLP-18
receptors. All other N-terminal alanine-substituted peptides induced right-shifts of the
concentration-response curve compared to the parent peptide on both receptors; this was most
evident for the substitution of P3 for alanine, Ala3 (Fig. 3A, B). The Ala4 modification,
DVPAVLRFa, also induced a significant loss of agonist potency for both receptors. On the other
hand, the Ala2 modification was similar to the parent peptide on NPR-4 (Fig. 3A), but induced a
shift similar to the Ala4 derivative on NPR-5a (Fig. 3B). All N-terminal alanine-substituted
peptides had the same maximum response as the parent peptide.
Figure 3. Concentration-response curves of FLP18-6 N-terminal alanine modifications on receptors expressed
in yeast. NPR-4 (A) and NPR-5a (B) were challenged with increasing concentrations of FLP18-6, DVPGVLRFa
(■), Ala1 AVPGVLRFa (●), Ala2 DAPGVLRFa (), Ala3 DVAGVLRFa (c) or Ala4 DVPAVLRFa (d). Growth in
CM/Leu-/His- media was measured by Alamar blue fluorescence. Data were normalized to the highest value and are
presented as the means and SEM of three experiments, each in triplicate.
C-terminal alanine modifications (sequential substitution of the 4 last residues in FLP18-6 by
alanine) showed stronger impacts on potency and maximum response than N-terminal alanine
91 modifications (Fig. 4A, B). The Ala5 derivative showed a more pronounced shift to the right in
the concentration-response curves for both receptors than Ala4. Ala5 elicited the same FLP18-6
Emax at NPR-4 but was less effective at NPR-5a: it only elicited 74% of the maximum response
induced by FLP18-6 on NPR5a (Fig. 4B). The Ala7 derivative was less potent on NPR-5a but
induced the same response as Ala5 on NPR-4. This alanine-substituted peptide showed a
reduction in the Emax to 70% on NPR-4 and 15% on NPR-5a compared to the parent peptide.
Surprisingly, the Ala6 derivative DVPGVARFa was less potent than Ala7 on NPR-4 and did not
activate NPR-5a at the highest concentration assayed. Alanine modification in the C-terminal
amino acid, Ala8, completely inactivated the peptide. None of the inactive alanine analogs, as
well as DPGVLRF-OH, had antagonist effects on the response to the parent peptide on both
receptors (not shown).
Figure 4. Concentration-response curves of FLP18-6 C-terminal alanine modifications on receptors expressed
in yeast. NPR-4 (A) and NPR-5a (B) were challenged with increasing concentrations of FLP18-6, DVPGVLRFa
(■), Ala5 DVPGALRFa (●), Ala6 DVPGVARFa (), Ala7 DVPGVLAFa (c) or Ala8 DVPGVLRAa (d). Growth in
CM/Leu-/His- media was measured by Alamar blue fluorescence. Data were normalized to the highest value and are
presented as the means and SEM of three experiments, each in triplicate.
EC50 values for FLP18-6 and analogs on NPR-4 and NPR-5a are summarized in Table 1. We
observed three patterns of activity for the FLP18-6 analogs: inactive, same potency as parent
peptide, change in potency, and change in both potency and Emax. In general, amide group
deletion and modification at F8 create inactive peptides; N-terminal alanine modifications affect
potency, especially the Ala3 substitution, while C-terminal substitutions affect both potency and
maximum response, especially at NPR-5a.
92 These patterns differ to some extent between the two FLP-18 receptors; for example, Ala2 is the
only peptide that has the same potency as the parent peptide on NPR-4, but is less potent on
NPR-5a (Fig. 3). Ala5 is a full agonist with reduced potency on NPR-4, but is a low potency,
incompletely effective agonist on NPR5a (Fig. 4). Peptides shorter than PGVLRFa as well as
Ala6 are low-potency agonists on NPR-4 but are not active on NPR-5a (Fig. 2 and 4). NPR-4
has a lower EC50 for the parent peptide, is able to recognize short peptides and C-terminal
substitutions at high concentrations, but is in general is more sensitive to alanine modifications
than NPR-5a.
NPR-4
FLP18-6 peptides
EC50 µM
C.I1 µM
NPR-5a
EC50s
Ratio2
EC50 µM
C.I1 µM
EC50s
Ratio2
DVPGVLRFa
0.48
0.44-0.52
1.93
1.80-2.08
PVGVLRFa
2.07
1.91-2.23
4.8
114
4
GVLRFa
3.52
3.29-3.76
8.2
ND
VLRFa
5.89
5.23-6.53
14
ND
DVPGVLRF-OH
ND3
ND
C-truncation
AVPGVLRFa
0.32
0.27-0.38
0.5
0.94
0.73-1.22
0.3
Ala1
DAPGVLRFa
0.70
0.62-0.80
1.1
11
3
Ala2
DVAGVLRFa
8.01
7.97-8.22
13
20
6
Ala3
DVPAVLRFa
4.00
3.99-4.02
6.6
11
3
Ala4
DVPGALRFa
36.6
26.9-49.6
78
38.5
37.4-39.6
8.9
Ala5
DVPGVARFa
111
236
ND
Ala6
DVPGVLAFa
28.7
18.4-44.4
61
75.3
69.6-81.6
17
Ala7
DVPGVLRAa
ND
ND
Ala8
Table 1. Analysis of concentration-response curves of FLP18-6 analogs tested on NPR-4 and NPR-5a. Data
from three experiments in triplicate were analyzed by a non-linear regression, sigmoidal – variable scope model,
using GraphPad software (GraphPad, San Diego, CA). 195% Confidence Intervals. 2EC50 ratio between analog and
parent peptide evaluated in the same experiments. EC50 values for the parent peptide differ in some cases due to
variations between experiments and clones. Despite this, comparisons with parent peptide are not affected. 3No
activation was detected at the highest concentration tested. 4Ambiguous EC50 value, as the high concentrations tested
did not generate a plateau.
Parent peptide
N-truncations
3.3.
FLP18-6 and analog effects in C. elegans cut preparations
Previous work has shown that FLP18-6 induces a curling phenotype in wt C. elegans cut
preparations (Ruiz-Lancheros et al., unpublished observations). This phenotype was consistent
between preparations with an onset time of 5 min when FLP18-6 was assayed at 100 μM. Cut
worms of the npr-5(ok1583) lf mutant strain did not show the wt curling phenotype upon
exposure to FLP18-6 under the same conditions, and the phenotype was recovered in the mutant
93 strain when NPR-5 was expressed extrachromosomally under its endogenous promoter. When
cut preparations of the npr-4(tm1782) lf mutant strain were tested in situ with FLP18-6, no loss
of the FLP18-curling phenotype was observed; suggesting the outcome of NPR-4 activation by
FLP18-6 does not affect locomotion in the same way as NPR-5 activation. Accordingly, we only
used wt and an npr-5 mutant strain to evaluate the effects of FLP18-6 analogs on C. elegans cut
preparations (Fig. 5). Due to the high concentration of peptide used to observe in situ effects in
the short time available for observation, this experiment was not designed to titrate each FLP186 analog, but only to compare differences among peptides.
Figure 5. Effects of FLP18-6 and analogs in C. elegans bioassay. Percentage of C. elegans cut worms from wild
type (WT) and npr-5 mutant npr-5(ok1583) strains that show FLP18-curling phenotype in presence of truncated
analogs of FLP18-6 and an alanine scan series. 10-12 cut worms per strain were tested with 100 μM peptide in APF.
Fisher’s exact test was used to detect significant differences between the percentage of wt cut-worms that showed
the curling phenotype in parent peptide and in FLP18-6 analogs. ***P<0.0001, **P<0.01, P*<0.05, or not
significant (n.s) compared with parent peptide.
None of the FLP18-6 analogs induced a curling-phenotype in npr-5 mutant cut preparations,
confirming that the loss of npr-5 expression prevents the locomotion effect induced by FLP-18
94 peptides (Fig. 5). In the in situ assay, very few C. elegans wt cut-worms showed the curling
phenotype when exposed to DPGVLRF-OH or Ala8, DVPGVLRAa. Responses were
significantly different than the response produced by the parent peptide in wt cut-worms,
suggesting that these peptides are inactive on NPR-5a as observed in the in vitro bioassay (Fig.
2B, 4B). The amidated peptide PGVLRFa (100 μM) induced the curling phenotype in wtworms, although a lower percentage of worms responded compared to the parent peptide. In
addition, the percentage of worms exhibiting the phenotype was lower when a shorter amidated
peptide (GVLRFa) was used. These observations are compatibles with results from in vitro
bioassay for NPR-5a (Fig. 2B). However, wt-cut worms exposed to the amidated tetrapeptide
VLRFa showed the curling phenotype even though this peptide was inactive in in vitro bioassay
for NPR-5a (Fig. 2B).
The C-terminal alanine modifications Ala6 and Ala7 produced a drop in the percentage of worms
that showed the curling phenotype compared with the parent peptide. The Ala5 peptide generated
the phenotype, but the percentage of responding worms was significantly lower than observed
with the parent peptide. In general, all C-terminal alanine modifications reduced the activity of
peptides in the in situ bioassay. In contrast, most of the N-terminal alanine modifications did not
change significantly the number of worms that showed the curling phenotype compared with the
parent peptide. The Ala3 peptide was the only N-terminal alanine analog that induced the curling
phenotype in a significantly lower percentage than the parent peptide. This peptide also showed a
high EC50 value and right-shifted the in vitro concentration-response curve for NPR-5a (Fig.
3B).
4.
Discussion
We investigated the interaction of DVPGVLRFa (FLP18-6) peptide with its receptors NPR-4
and NPR-5a in SAR studies, aiming to understand the basis of the ligand-receptor interactions
and inform the search for non-peptide ligands as antiparasitic drug candidates. The flp-18
precursor gene is highly conserved in free-living nematodes and gastrointestinal and filarial
parasitic nematodes (McCoy et al., 2014). flp-18 is widely expressed in C. elegans and A. suum
neurons, although with different expression patterns (Cohen et al., 2009; Nanda and Stretton,
95 2010; Stawicki et al., 2013). Despite localization differences, physiological studies in A. suum
and C. elegans, as well as flp-18 orthologs silencing in G. padilla, indicate that FLP-18 peptides
have roles in the neuromuscular system across the phylum Nematoda (Fellowes et al., 2000;
Davis and Stretton, 2001; Kimber et al., 2007).
Furthermore, flp-18 overexpression in C. elegans produces uncoordinated movement and
increased curvature. Induced overexpression of flp-18 in cholinergic motor neurons decreases the
activity level of the locomotory circuit (Rogers et al., 2003; Stawicki et al., 2013). These suggest
that FLP-18 peptides control curvature and have homeostatic effects on the excitation-inhibition
pattern which governs movement. The curling phenotype we observed in wt cut-worm
preparations upon exposure to FLP18-6 is compatible with the effects seen in overexpression
experiments.
C. elegans reverse pharmacology studies and loss-of-function studies have demonstrated that
FLP-18 peptides are associated with receptors NPR-1, NPR-4 and NPR-5 (Rogers et al., 2003;
Kubiak et al., 2007; Cohen et al., 2009). Although these associations have not been established in
parasitic nematodes, the three receptors are highly conserved across the Nematoda (McCoy et al.,
2014). The conservation of receptors, together with the well-characterized physiological
activities of FLP-18 peptides, makes these receptors good anthelmintic targets and legitimizes
studies of their biological roles and interactions with their endogenous ligand.
Heterologous expression of FLP-18 receptors has shown that NPR-4 activation couples with the
Gαi/o signaling cascade in Xenopus oocytes, while NPR-5 interacts with Gαq in both mammalian
cells and Xenopus oocytes (Kubiak et al., 2007; Cohen et al., 2009). We observed conservation
of these signaling cascades in the yeast expression system, suggesting that the receptor/G-protein
interaction is maintained in different heterologous expression systems, as observed for the flp-2
receptor (Larsen et al., 2013). The yeast system provides a sensitive and simple way to detect
agonists and is suitable for SAR studies. It is also well-suited for screening for non-peptide
GPCR agonists and antagonists as potential anthelmintics (Geary et al., 2012; Geary and
Ubalijoro, 2012).
96 Although we associated FLP18-6 with both receptors in the in vitro bioassay, we observed in an
in situ C. elegans assay that the npr-5 mutant lost the FLP18-curling phenotype observed in wt
worms, while an npr-4 mutant strain retained the phenotype. This result suggests that the
interaction of FLP18-6 with NPR-5 accounts for the locomotor effects of the FLP-18 peptides.
Cohen et al. (2009) observed that the npr-5p::rfp transgene is expressed in body muscle, neck
and head, as well as in some amphidial neurons, interneurons (AIA and AUA) and phasmids.
Stawicki et al. (2013) reported a similar expression pattern and suggested that peptides derived
from flp-18 overexpression in cholinergic neurons can act directly on the muscle to inhibit
contraction or promote relaxation. These effects are consistent with our in situ findings in cut
worms. Moreover, expression of the npr-4p::rfp transgene is restricted to the nervous system,
coelomocytes, parts of the intestine and rectal gland cells (Cohen et al., 2009). NPR-4 has minor
effects on the excitatory-inhibitory balance in the locomotory circuit (Stawicki et al., 2013), and
probably has only limited direct roles in the neuromuscular system. This evidence and the lack of
NPR-4 expression in muscle explain the retention of the FLP18-curling phenotype in the npr-4
mutant strain in in situ assay. Despite this, strong evidence supports the role of NPR-4 activation
by FLP-18 in the modulation of food-related behaviors, such a fat metabolism, chemosensation
and foraging, which are also essential in worm biology (Cohen et al., 2009; Frooninckx et al.,
2012) but which would not be detected in the cut-worm assay.
Our FLP18-6 SAR analysis on NPR-4 and NPR-5a in vitro and in situ confirms the general
importance of the C-terminal amide group for activity of the FLP family (Geraghty et al., 1994;
Bowman et al., 1996; Bowman et al., 2002). In addition, our results show that the N-terminal
motif (DV-) in FLP18-6 is not essential for receptor activation. Kubiak et al. (2007) observed
that short peptides encoded in flp-18 are better NPR-5a agonist than longer peptides, which
supports our findings. In the yeast expression system, PGVLRFa was the minimal sequence
required for NPR-5a activation, while shorter amidated peptides were able to elicit responses on
NPR-4. SAR analysis of other FLPs has shown different roles of peptide N-termini in activity.
For example, all N-terminal truncations in KNEFIRFa (AF1) disrupted contraction of A. suum
body muscle compared to AF1 (Bowman et al., 1996), while N-terminally truncated forms of
SDPNFLRFa (PF1) relaxed denervated A. suum body muscle to the same extent as PF1
97 (Bowman et al., 2002). Results with FLP18-6 N-terminally truncated analogs also suggest that
P3 is critical for normal NPR5a activation, but is not as important for NPR-4 recognition. The
amidated tetrapeptide VLRFa activated NPR-4 at high concentrations but with a lower Emax
compared with GVLRFa and the parent peptide, which suggests that the pentapeptide is likely
the minimal core for NPR-4 full activation. In situ, we observed the canonical FLP-18 curling
phenotype in wt cut-worms exposed to VLRFa, but not in the presence of GVLRFa. In general,
amidated tetrapeptides or shorter peptides seem to be inactive in in situ or in vitro bioassays;
however, it is not possible to predict if the tetrapeptide can also act in situ on GPCRs that
respond to peptides with the same C-terminus.
To evaluate the contribution of each FLP18-6 residue to receptor activation in vitro and in situ,
we used an alanine scan series in which each residue was sequentially replaced by alanine.
Alanine has a small non functional side chain and does not introduce significant changes in
peptide secondary structure (Bowman et al., 1996; Gregoret and Sauer, 1998); thus, an alaninescan series can give information of the effects of side chain and position in the activation of
receptors (Weiss et al., 2000). All alanine substitutions affected NPR-4 and NPR-5a activation,
and the alanine position differentially influenced the peptide interaction with each receptor. In
vitro and in situ results with the Ala6, Ala7 and Ala8 substitutions confirmed that side chains at
the C-terminus are important for receptor activation, as was observed in SAR studies for PF1 and
AF1 (Bowman et al., 1996; Bowman et al., 2002). Our results suggest that L6 and F8 have a
higher impact on receptor activation than R7, which is not the case for PF1 and AF1. A recent
SAR analysis of DGYRPLQFa (NLP-12) suggested that the side chain of the penultimate basic
residue (Gln for NLP-12 and Arg for FLP-18) stabilizes the backbone of the peptide rather than
interacting with the receptor (Peeters et al., 2012); this could also be the case for R7 in FLP-18
peptides. On the other hand, the Ala1 substitution, AVPGVLRFa, was a more potent agonist of
both receptors in vitro. This peptide modification generated the known FLP AF3 (A. suum),
which is a more potent agonist than DVPGVLRFa of NPR-5 in mammalian cells (Kubiak et al.,
2007). As mentioned above, the FLP18-6 N-terminus seems to be irrelevant for receptor
activation; it is possible that elimination of the side carboxyl group in D1 facilitates the
accessibility of the peptide to its site of action in NPR-5a and NPR-4, resulting in a better
98 agonist. Unfortunately, the high concentrations needed in the in situ assay did not permit us to
easily distinguish agonist potency in this model.
The alanine scan analysis also revaeled that P3 has an essential role in the activation of both
receptors. Although NPR-4 recognizes shorter amidated peptides lacking this proline, the Ala3
substitution induced a clear reduction in potency on NPR-4. It is possible that the low potency of
the Ala3 substituted peptide on both receptors is due to the lack of interaction of the proline side
chain with receptors. A similar alanine for proline substitution in PF1 does not have an effect on
activity; however, only peptide structural studies will establish if changes in secondary structure
produced by this substitution are responsible for the change in peptide activity. In addition, our
results indicated that V5 is also important for activation of both receptors. The Ala5 substituted
peptide has low potency as an agonist at both receptors and elicited the same maximum response
as parent peptide at NPR4 but not at NPR5, which indicates a difference in the manner in which
these receptors recognize the V5 side chain. Moreover, the sensitivity to alanine modifications
detected for NPR-4 and NPR-5 activation in vitro also differed. The peptide potency at NPR-4
changed significantly with alanine modifications compared to NPR-5. These observations,
combined with the ability of NPR-4 to recognize short peptides, suggests that access to the NPR4 binding site may be more open than in NPR-5, but that NPR-4 interacts more with amino acid
side chains compared to NPR-5. We hypothesise that NPR-4 and NPR-5 may differ at least
slightly in the FLP binding site, which should be proved with binding studies and the modelling
of receptor tertiary structure using a rhodopsin-based or invertebrate-GPCR model (Mugumbate
et al., 2011).
In addition to these insights into the FLP18-6 interaction with its receptors, our SAR analysis
informs attempts to use in silico approaches to identify non-peptide agonists that act on both
receptors. In silico studies should focus on non-peptide mimetics that conserve the chemical core
of the FLP-18 residues important for receptor activation, including those residues that interact
differentially with both receptors. We hypothesise that a potent NPR-4 and NPR-5 agonist
should contain a chemical group that mimics the prolyl moiety of P3 and a group that mimics the
position and aliphatic side chain of V5. Since the FLP18-6 C-terminus is necessary for receptor
activation, a non-peptide agonist should also have a group that mimics the chemistry of L6, F8
99 and the C- terminal amide. Additionally, replacement of aspartate with an uncharged moiety
(D1) may improve agonist potency. These predictions are not exclusive; and only performing in
silico studies can support or refute these proposals.
Acknowledgments
This work was supported by funds from NSERC, the Canada Research Chairs program, the Bill
and Melinda Gates Foundation, CIHR and the Grand Challenges Canada program (to T.G.G.).
E.R.L. was supported by a Tomlinson Fellowship and the FQRNT Centre for Host-Parasite
Interactions. We thank J. Broach (Cadus, Inc., Princeton, NJ) and Pfizer Animal Health (now
Zoetis, Inc., Kalamazoo, MI) for providing S. cerevisiae strains. We also thank the
Caenorhabditis Genetics Center and the National Bioresource Project for the Nematode for
provision of strains.
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Nematode infections still affect a high percentage of the world population, especially in low
income countries where health resources and infrastructure are limited. They also affect small
companion animals and are of great concern in livestock and crop production. Unfortunately,
resistance to most chemotherapeutic agents in livestock and domestic animals has appeared and
there is a high potential of resistance in humans (Vercruysse et al., 2011). Limitations of the
available drugs, the high dissemination of some infections, current drug-use patterns that favor
the selection of resistance alleles, and the use of drugs with similar molecular targets, have
inevitably contributed to resistance and will continue to do so in the future (Martin and
Robertson, 2010; Vercruysse et al., 2011). Clearly a better understanding of anthelmintic
pharmacology as well as new molecular targets and new drugs that escape existing mechanisms
of resistance are urgently needed (Geary et al., 2010).
C. elegans has been a useful model nematode to investigate the mode of action of different
anthelmintics. Thanks to its general similarities with parasitic nematodes, its experimental
tractability and the genetic resources available for this free-living nematode, it has also served
well for the study of anthelmintic pharmacology and the mechanisms of resistance in parasitic
species. In addition, C. elegans can be useful in identifying and characterizing new molecular
drug targets and for anthelmintic discovery (Holden-Dye and Walker, 2012). In this work, we
standardized a C. elegans cut-worm model that gave insights into the pharmacology of the new
anthelmintic derquantel and helped in the deorphanization and characterization of FLP-GPCRs.
Most current anthelmintics have an identified target in C. elegans and several have a direct effect
in intact specimens; however derquantel appears to be inactive in C. elegans despite its potency
against H. contortus and other trichostrongylid parasites. Using the cut-worm model
(Manuscript I), we observed that derquantel is able to paralyze C. elegans cut-preparations and
antagonizes nAChR agonists like bephenium and nicotine. These results suggested that the
derquantel molecular target is present in C. elegans and is an nAChR, as is predicted in parasitic
species. Additionally, using C. elegans cut-preparations it was observed for the first time that
103 derquantel does not interfere with the effects of the new anthelmintic monepantel, an nAChR
agonist. This suggests that these new drugs may not suffer from a negative interaction if used in
combination to treat nematodiases.
The fact that derquantel has an effect in cut-preparations but not in intact C. elegans specimens,
suggests that the cuticle is a barrier to its entry, which could explains its poor activity in intact
worms. Results also suggest that the C. elegans cuticle may be more restrictive to permeability
than the cuticle in parasitic species that respond to derquantel. This agrees with the low
bioaccumulation levels of small molecules in C. elegans observed by Burns et al. (2010). These
observations call into question the use of intact C. elegans specimens for anthelmintic screening
without pre-selection of compounds or neglecting the cuticle as a barrier. As a result of our
observations, the cut-worm model is currently used in our laboratory to re-screen drugs that
appeared inactive in intact C. elegans worms and to test new drug-like compounds; it is also used
in the in situ characterization of the C. elegans derquantel molecular target. Due to the technical
limitations in dissecting worms, the cut worm model cannot be used for high-troughtput screens
for potential anthelminctis or other drugs; instead, the use of permeability mutant C. elegans
strains should be considered. In this regard, the glycosyltransferase bus-8 and the epimerase bus5 mutants have shown increased permeability to drugs with low bioacummulation in wild type
specimens (Gregoret and Sauer, 1998).
Like derquantel, neuropeptides are inactive in C. elegans specimens because their large size and
hydrophilic nature limits their accessibility through the cuticle. Using the cut-worm model, it
was observed that selected FLPs (which are potent in A. suum physiological assays) induce a
range of locomotor effects (phenotypes) in C. elegans cut preparations. These novel observations
prompted the use of the cut-worm model to identify endogenous FLP-GPCRs in situ
(Manuscript II). Cut preparations from C. elegans strains with loss-of-function mutations in
candidate GPCR genes were interrogated for the retention of a particular FLP-phenotype
observed in wt cut-worms. Validation experiments using the peptide DVPGVLRFa (FLP18-6)
and its receptor NPR-5 suggested that the strategy was robust enough to pair (deorphanize) FLPs
with GPCRs, and that associations found in situ are reliable.
104 Although this in situ deorphanization strategy in cut preparations cannot be scaled, it allows
prioritization of a search for receptors for those FLPs that have profound effects in the
neuromuscular system. Likewise, the pre-selection of candidate receptors using bioinformatic
tools narrows the number of receptors to screen in situ. On the other hand, the strategy cannot be
used in parasitic nematodes since it is not easy to get a significant number of specimens in most
cases and there are no mutants available. Nevertheless, considering that homologs of C. elegans
FLPs and FLP-GPCRs are present across the nematode Phyla (McVeigh et al., 2005; McCoy et
al., 2014), results from in situ deorphanization in C. elegans may be extended to parasitic
nematodes.
One of the advantages of using C. elegans is the availability of a high number of mutant strains.
However, the use of knockout strains can be controversial if the null mutation of a specific gene
is not achieved and if the KO strains carry other unknown mutations. In the in situ study, null
mutations in selected GPCR genes were confirmed, and the inclusion of multiple FLPs and
strains controlled for the presence of additional mutations that could affect the phenotypes
observed in the cut-worm bioassay and consequently the FLP-GPCR associations. Additionally,
the recovery of the wt FLP18-phenotype in an npr-5 KO strain by introducing the transgene
pnpr-5::npr-5, suggested that the ligand-receptor interaction accounts for the locomotor effects
induced by the peptide and are not the results of other mutations. Current efforts in our
laboratory to target the rescue of FLP-phenotypes in mutant strains that carry mutations in in situ
deorphanized receptors are underway. RNAi methods (Ahringer, 2006) and the CRISPR RNAguided nuclease system (Weiss et al., 2000) will be explored to reinforce the results observed in
KO strains.
Through in situ screening of receptors using the cut-worm model, 9 novel C. elegans FLP-GPCR
pairs were identified; an impressive achievement compared with the total of 12 matches
identified in reverse pharmacology studies during the last 15 years. The in situ strategy also
identified pairs that were undetected in heterologous receptor expression platforms in which
several receptors were expressed and a high number of peptides tested. Clearly our strategy
accelerates the deorphanization process and provides invaluable data. Since we identified GPCRs
for physiologically relevant FLPs, it is expected that these receptors play key roles in
105 neuromuscular functions and may be promising targets for new anthelmintics. For instance, we
identified FRPR-18 (FMRFamide peptide receptor-18 encoded by T19F4.1 gene), as the
endogenous receptor for peptide KHEYLRFa (AF2), which appears the most abundant peptide in
A. suum, H. contortus and P. redivivus and has potent inhibitory effects in A. suum and H.
contortus neuromusculature (Mousley et al., 2010). FRPR-18 has also been associated with FLP2 peptides (-EPIRFa), but locomotor effects induced by these peptides were not observed in situ,
as was observed with AF2. This situation may indicate that in vitro ligand-receptor association
may not imply in situ interaction, and receptor localization and peptide release in its proximity
determine the in situ association. Whether this is the case for the FLP-2/FRPR-18 and
AF2/FRPR-18 pairs remains to be verified with the elucidation of FRPR-18 and peptides
expression patterns and biological roles.
Results from in situ studies guided the selection of receptors to study pharmacologically in
heterologous systems. In the yeast recombinant system, it was observed that AF2 activates
FRPR-18 in a concentration-dependent manner and that the system couples with the Gαqsignalling cascade (Manuscript II). Similar results were observed for FLP-2 peptides
(Manuscript IV). Additionally, the yeast system served to express the FLP-18 receptors NPR-5
and NPR-4 (Manuscript III). These data indicate that yeast can be used for functional
expression of selected C. elegans GPCRs and to screen compounds that act as agonists or
antagonists of these receptors.
As a first time, the three receptors FRPR-18, NPR-5 and NPR-4, expressed in the yeast system,
have been used by our laboratory to screen for non-peptide ligands agonist or antagonist in
libraries of African natural products, fermentation and marine products, and synthetic compound
libraries. The objective is to find promiscuous ligands that act in more than one receptor and
scape receptor-mediated mechanisms of drug resistence, consecuently the agonist screnning
assay has been peformed multiplexing the different yeast strains expressing receptors using as
control for selectivity the D. melanogaster allatostatin receptor (Geary, 2012) (Geary et al.,
2012; Geary and Ubalijoro, 2012). Finding agonists have been harder than finding antagonists
maybe because there are more binding sites that can disrupt receptor function than those that can
106 activate it (Geary, 2012). Currently our laboratory has identified more than 50 FLP-GPCR
antagonists that deserve further validation as putative anthelmintics.
The yeast expression system was not suitable to study the pharmacology of all in situ
deorphanized receptors. Some recombinant GPCRs were not activated by the corresponding FLP
even though the cellular expression and membrane localization of receptors in yeast were
documented. Considering this, we must ask if a successful FLP-GPCR association in situ implies
a direct ligand-receptor interaction or if the in situ system was susceptible to produce false
positives. Certainly more work is needed to conclude that the lack of in vitro receptor activation
indicates no association between a specific ligand-receptor pair. For example, the lack of
activation for some GPCRs in the yeast system maybe due to the lack of co-expression of C.
elegans factors needed for functional expression, and/or inadequate coupling of recombinant
GPCRs with the endogenous yeast signalling cascade. Similar problems have been encountered
in heterologous expression of other receptors, including nematode FLP-GPCRs, in other systems
(Dunham and Hall, 2009). It is also possible that neuropeptide receptors must form heterodimers
in vivo to be functional, or perhaps signal through G-protein-independent mechanisms (Levoye
and Jockers, 2008). Whether nematode neuropeptide GPCRs have these characteristics is
unknown.
It is possible that in situ effects of FLPs in cut-worms reflect actions of indirect modulation of
GPCRs by others from the same or different signalling cascade giving as a result an in situ false
positive direct association. New evidence suggests that neuropeptides can act extra-synaptically
as neuromodulators, and different GPCRs activated by the same neuropeptide can play roles in
the same or different pathway by acting in different neurons or on different target neurons
(Cohen et al., 2009; Frooninckx et al., 2012; Stawicki et al., 2013). Additionally neuropeptides
from different families can be involved in the same feedback signalling pathway activating
different GPCRs to finally induce the same outcome behavior (Chalasani et al., 2010). In this
case, a mutation in any of the GPCRs that interrupts the neuropeptides feedback has the same
effect on the neural circuit and can be misleading as direct ligand-receptor association.
Accordingly, to further investigate whether the effects of FLPs in cut worm were due to direct or
indirect ligand-receptor activation additional approaches should be used; like, phenotypes
107 characterization of peptide/receptor knockdowns and overexpression, phenotype rescue
experiments and correlation of expression and biological roles of both ligand and receptor.
Nevertheless, the difficulties we encountered in the functional expression of FLP-GPCRs in the
yeast system illuminates the limitations of heterologous expression systems, and questions their
reliability to confirm ligand-receptor associations, as well as their use in high-throughput
expression of receptors to screen for endogenous or non endogenous agonists or antagonists. Our
observations support recent efforts to deorphanize GPCRs in situ using RNAi-mediated
approaches in parasitic helminths (Zamanian et al., 2012; Atkinson et al., 2013). These
approaches cannot provide proof for direct ligand-receptor interactions and have limitations for
pharmacological studies, but they are useful in a first screen for receptors and in combination
with the strategies mention above can help in the deorphanization process.
On the other hand, using the in situ deorphanization strategy, it was observed that one FLP can
act as a ligand of more than one GPCR, and several GPCRs can be associated with the same
FLP. This promiscuity in the neuropeptidergic system has been documented in other
deorphanization studies and in neuropeptide physiological studies in other invertebrates. There is
insufficient structural information on nematode neuropeptides and their receptors to predict
ligand-receptor interactions and the mechanisms that govern multiple associations. Aiming to
understand the basis for these ligand-receptor interactions, we performed structure-activity
relationship studies for the peptide DVPGVLRFa (FLP18-6) using the cut-worm model and the
FLP-18 receptors NPR-4 and NPR-5 heterologously expressed in yeast, (Manuscript III).
Although the in situ bioassay did not allow us to easily distinguish agonist potency, and in situ
results may only account for the NPR-5 interaction with FLP18-6, it was observed in situ that the
peptide C-terminus has an essential role in activity and that specific side chains in the peptide Cterminus determine receptor activation. In vitro studies reproduced these observations and also
suggested a differential interaction of the FLP18-6 C-terminus with each receptor. Slight
differences in the manner in which NPR-4 and NPR-5 interact with FLP18-6 side chains at the
molecular level are predicted based on these in vitro results. Currently we are performing in vitro
studies for the Brugia malayi NPR-4 and NPR-5 homologs in order to confirm the conservation
108 of the ligand – receptors interaccion between species and validate the use of C. elegans receptors
for screen for anthelmintics candidates.
The information provided by the structure-activity relationship studes is useful in the
confirmation of the ligand binding site in both receptors and to model ligand pharmacophores
that guide the in silico rational search of promiscuous non-peptide ligands that target FLP-18
receptors and can interfere with FLP-18 signalling pathways in parasitic nematodes. Both in situ
and in vitro bioassays can be used to test candidate ligands that mimic the chemical space of
FLP-18 (shape, charge, lipophilicity and the chemistry of residues important for activation) and
can interact with NPR-4, NPR-5 and other FLP-GPCRs.
At the outset of this PhD thesis, we standardized a cut-worm model that has been useful in
expanding knowledge on the derquantel mode of action and in deorphanizing FLP-GPCRs that
are promising targets for new anthelmintics. It also helped to elucidate ligand-receptor
interactions that can inform attempts to use in silico approaches to identify non-peptide agonists
as chemotherapeutic agents. Results with the cut-worm model have also opened avenues for its
use in future characterization of anthelmintic targets and in screens of drug-like compounds with
better success than screens using intact C. elegans specimens. It is expected that the novel FLPGPCR associations identified in situ using the cut-worm model will guide future studies that
validate these associations in parasitic nematodes, characterize pharmacologically and
functionally the novel deorphanized receptors, and discover novel anthelmintics that target
neuropeptide GPCRs.
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mediated approach to G protein‐coupled receptor deorphanization: proof of principle and characterization of a planarian 5‐HT receptor. PloS one 7, e40787, doi:40710.41371/journal.pone.0040787. 111 Appendix. Manuscript IV.
Functional expression and characterization of the C. elegans G-protein-coupled FLP-2 receptor
(T19F4.1) in mammalian cells and yeast
Martha J. Larsen1,a, Elizabeth Ruiz-Lancheros2, Tracey Williams1, David E. Lowery1,b, Timothy
G. Geary1,c, Teresa M. Kubiak1
1
Pfizer Animal Health Discovery Research, Veterinary Medicine Discovery Research,
Kalamazoo, MI 49001, USA 2Institute of Parasitology, McGill University, 21,111 Lakeshore
Road, Ste-Anne de Bellevue, Quebec H9X 3V9, Canada.
Present addresses: aLife Sciences Institute, University of Michigan, 210 Washtenaw Ave. Ann
Arbor, MI 48109. bNovartis Animal Health, 3200 Northline Ave., Greensboro, NC 27408-7611.
c
Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Ste-Anne de Bellevue,
Quebec H9X 3V9, Canada.
Published in: International Journal for Parasitology: Drugs and Drug Resistance,
3:1-7, doi: 10.1016/j.ijpddr.2012.10.002
(2013)
112 Abstract
Profound neuropeptide diversity characterizes the nematode nervous system, but it has proven
challenging to match neuropeptide G protein-coupled receptors (GPCR) with their cognate
ligands in heterologous systems. We have expressed the Caenorhabditis elegans GPCR encoded
in the locus T19F4.1, previously matched with FMRFamide-like peptides encoded on the flp-2
precursor gene, in mammalian cells and in the yeast Saccharomyces cerevisiae. Pharmacological
characterization revealed that the receptor is potently activated by flp-2 peptides in CHO cells (~
10 nM EC50) and in yeast (~100 nM EC50), signaling through a Gαq pathway in each system. The
yeast GPCR expression system provides a robust assay for screening for agonists of the flp-2
receptor and is the target of an ongoing high-throughput screening exercise.
113 1.
Introduction
FMRFamide-like peptides (FLPs) are important neurotransmitters and neuromodulators in
invertebrates, including parasitic helminths, arthropods and other organisms (Price and
Greenberg, 1989; Greenberg and Price, 1992; Walker, 1992; Halton et al., 1994; Shaw C et al.,
1996; Brownlee et al., 2000; Maule et al., 2002). At present, nearly 80 distinct FLPs encoded on
34 flps (precursor genes) have been predicted from the Caenorhabditis elegans genome
(McVeigh et al., 2005; McVeigh et al., 2006; Husson et al., 2007; Li and Kim, 2008; Walker et
al., 2009; Li and Kim, 2010). This number could be higher; despite extensive bioinformatic
searches, all family members may not have been identified. In addition to FLPs, 48 C. elegans
neuropeptide-like protein (nlp) genes have been identified, defining almost 140 total putative
neuropeptides in this classification (Nathoo et al., 2001; McVeigh et al., 2006; Husson et al.,
2007; Li and Kim, 2008; McVeigh et al., 2008; Li and Kim, 2010), as well as 40 precursor genes
encoding insulin-like peptides (Li and Kim, 2008; Li and Kim, 2010).
Approximately 60 genes encoding putative neuropeptide G protein-coupled receptors (GPCRs)
have been annotated in the C. elegans genome (Bargmann, 1998; Keating et al., 2003). The
number of putative flp- and nlp-encoded neuropeptides is much higher than the number of
predicted neuropeptide GPCRs, implying that a single receptor is likely to be activated by
multiple peptides. In C. elegans (Nathoo et al., 2001; McVeigh et al., 2005; McVeigh et al.,
2006; Husson et al., 2007; Li and Kim, 2008; McVeigh et al., 2008; Walker et al., 2009; Li and
Kim, 2010) and other invertebrates (Bellés et al., 1999; Fujisawa et al., 1999; Furukawa et al.,
2001), multiple forms of related peptides are typically encoded on one precursor protein gene
and these peptide families can recognize the same GPCR, as has been shown for the Drosophila
allatostatin type A receptor (Larsen et al., 2001; Lenz et al., 2001) and as appears to be the case
for several FLP families analyzed in Ascaris suum physiological experiments (see McVeigh et
al., 2006). Since the publication of the C. elegans genome in 1998 (Consortium, 1998), a
relatively small number of orphan C. elegans neuropeptide GPCRs has been matched with a
cognate ligand (see Lowery et al., 2003; McVeigh et al., 2006; Husson et al., 2007)). Although
many orphan vertebrate GPCRs have been paired with endogenous ligands using approaches
employing heterologous receptor expression systems and reverse pharmacology (Civelli et al.,
114 2001), this approach has not been uniformly straightforward for nematode neuropeptidergic
GPCRs.
As part of a large-scale project devoted to identifying peptide-receptor matches in C. elegans
(Greenwood et al., 2005; Woods et al., 2010), we identified the GPCR annotated as T19F4.1 as a
receptor for peptides encoded on the flp-2 precursor gene (Lowery et al., 2003). This finding was
also reported by another group (Mertens et al., 2005). We report here further characterization of
the pharmacology of this putative C. elegans GPCR with its cognate ligands following
expression in mammalian cells and in the yeast Saccharomyces cerevisiae, which has proven
useful for the heterologous expression of GPCRs and for high-throughput screening for small
molecule ligands of these receptors (Broach and Thorner, 1996; Pausch, 1997; Ladds et al.,
2005; Minic et al., 2005; Wang et al., 2006; Dowell and Brown, 2009; Geary, 2012; Geary and
Ubalijoro, 2012). This GPCR has been termed the FLP-2 receptor (FLP-2R), because the
receptor activating peptides are encoded on the C. elegans precursor gene flp-2 (Mertens et al.,
2005).
2.
Materials and methods
2.1.
Materials
Synthetic peptides were made at Auspep Pty. Ltd. (Parkville, Australia) and Sheldon
Biotechnology Centre (McGill University). Chinese hamster ovary cell line CHO-10001A (CHO
cells), cell culture media, transfection and assay reagents were as described previously (Larsen et
al., 2001; Kubiak et al., 2002). U-73122 (a phospholipase C inhibitor) was obtained from the
Pfizer compound collection (Bleasdale et al., 1990). Yeast strains and vectors (Wang et al.,
2006) were obtained under license from Cadus Corp.
2.2.
Cloning and plasmid preparation
Molecular biological techniques followed either manufacturer’s recommendations or general
protocols. A variety of PCR primers were designed using the coding sequence for locus T19F4.1
as predicted in Wormpep (release 13). Bioinformatic analyses of the C. elegans genome using
previously cloned C. elegans FLP GPCRs (Kubiak et al., 2002; Kubiak et al., 2003) had
115 identified this gene as a candidate neuropeptide GPCR (not shown). The only significant
modification to the amplicon was the addition of an optimized translational initiation sequence
immediately preceding the authentic initiation codon (“GCC GCC”) (Kozak, 1987). Using
cDNA prepared from C. elegans strain N2 with sense and antisense primers deduced from the C.
elegans genome, PCR products encompassing the complete open reading frame of T19F4.1 were
cloned directly into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad, CA).
Nucleotide sequence analysis by standard protocols revealed the presence of 3 distinct clones,
each in multiple copies (Fig. 1). The longest of these was chosen for further analysis; this clone
was designated the Ce50b (flp-2R)/pCR3.1 plasmid and the cDNA it encodes is identical to
T19F4.1b (Lowery et al., 2003; Mertens et al., 2005).
2.3.
Cell culture, cell transfection and intracellular Ca+2 mobilization assay
CHO cells were cultured and transfected essentially as described (Kubiak et al., 2002; Kubiak et
al., 2003) with some modifications. The flp-2R/pCR3.1 plasmid (5 μg DNA/10 cm plate) was
used for transfection with the LipofectAmine PLUSTM (Invitrogen, Carlsbad, CA) method. The
assay for functional expression employed a 96-well fluorescence imaging plate reader (FLIPR)
(Molecular Devices, Sunnyvale, CA) essentially as described (Kubiak et al., 2002; Kubiak et al.,
2003), exposing the transformed cells to ~200 invertebrate neuropeptides (Kubiak et al., 2002).
In temperature shift experiments, transfected cells were split and plated in 96-well FLIPR plates
24 hr post transfection, followed by additional 24 hr of incubation at 37°C and then 24 hr 28°C
incubation prior to peptide testing. In some experiments, the phospholipase C inhibitor U-73122
was added in HBBS/HEPES/ probenecid, 0.1% DMSO, 10 μM final concentration, 10 min
before the addition of peptide.
2.4.
Expression in yeast
The ORF from plasmid flp-2R/pCR3.1 was subcloned into the yeast vector Cp4258, which
carries a LEU2 selectable marker (see Wang et al., 2006) following PCR amplification using 5’and 3’- terminal primers that generated an NcoI and an XbaI restriction site, respectively, which
were the cloning sites in the vector. The resultant flp-2R-Cp4258 plasmid was transformed into
E. coli for propagation using ampicillin selection and then transformed into a collection of strains
116 of S. cerevisiae based on CY13193 (MATα PFUS1-HIS3 far1Δ1442 gpa1Δ1163 his3 leu2 lys2
trp1 ura3 sst2Δ2 ste14::trp1::LYS2 ste18γ6-3841 ste3Δ1156 tbt1-1) as described (Wang et al.,
2006; Kimber et al., 2009). Each strain contains an integrated modified copy of the yeast Gαsubunit gene (GPA1) that incorporates the terminal pentapeptide sequences from the mammalian
Gi-, G12-, G13-, Gz-, Gq- and Gs-α proteins, in addition to others based on C. elegans G-α proteins
(gpa-1, gpa-2, gpa-7, gpa-9, gpa-12 and gpa-16), to facilitate the interaction between this protein
and heterologous GPCRs. It should be noted that the C-terminal pentapeptide is identical in C.
elegans and mammals for Gq (egl-30) and Gs (gsa-1). The yeast strains express HIS3 under
control of the FUS1 promoter when an agonist activates the heterologously expressed GPCR.
Thus, activation of FLP-2R by its peptide ligand leads to the correction of the histidine
auxotrophy, enabling growth in histidine-deficient medium.
Strains were cultured in Complete Minimal (CM) dropout medium (Sigma-Aldrich, St. Louis,
MO) and transformed using the lithium acetate method (Gietz et al., 1995). Positive
transformants were selected in plates with CM medium lacking leucine (CM/Leu-) and used for
receptor activation assays in CM liquid medium lacking leucine and histidine (CM/Leu-/His-),
supplemented with 0.05 M MOPS, pH 6.8. Briefly, a colony was grown overnight in CM/Leu- at
250 rpm and 30°C, cells were harvested by centrifugation at 12000 rpm for 1 min and rinsed
thoroughly in CM/Leu-/His- to eliminate histidine. Cell density was established by absorbance at
600 nm and 3000 cells/well in CM/Leu-/His- were seeded a 96-well plate to incubate with serial
dilutions of FLP-2 peptides. After 44 h incubation at 30°C, cell growth was measured by Alamar
Blue fluorescence after 2-4 h of incubation with 20 µl Alamar Blue (Sigma-Aldrich) at 30°C for
color development (Klein et al., 1997). Fluorescence was measured in a Synergy H4 Hybrid
Multi-Mode Microplate Reader (BioTek) set at 560 nm excitation/590 nm emission. Experiments
were performed three times in three replicates using as controls untransformed strains and
transformants in presence of histidine.
117 2.5.
Data analysis
Concentration-response curves for CHO cell and yeast experiments were analyzed by nonlinear
regression using Prism (GraphPad Software, Inc. San Diego, CA) based on each treatment run in
triplicate and expressed as mean values ± 95% confidence intervals (C.I.).
3.
Results
3.1.
Molecular cloning of T19F4.1/FLP-2R and phylogenetic analysis
Previous work described 2 alternatively spliced transcripts are encoded by the gene T19F4.1
(Mertens et al., 2005). PCR of C. elegans cDNA recovered the longer of these transcripts
(T19F4.1b), which was used for the work in this paper. Two other novel transcripts were also
detected that are distinct in the C-terminal region (Fig. 1). Examination of genomic sequence
suggested these transcripts were similarly generated via alternative splicing. Previous
phylogenetic analysis showed that this receptor is grouped with other known and putative C.
elegans neuropeptide GPCRs, including C26F1.6, which is activated by relatively high
concentrations of FLPs encoded on flp-7 and flp-11 (Mertens et al., 2004). The T19F4.1b cDNA
was used for all experiments reported here.
3.2.
Receptor expression and identification of cognate ligands for FLP-2R in mammalian
cells
The receptor-ligand matching was achieved by functionally expressing FLP-2R in CHO cells in
a transient transfection mode. A Ca2+ mobilization assay was used to monitor receptor activation
by FLIPR in response to > 200 synthetic peptides representing a variety of C. elegans and other
invertebrate FLPs. The expressed receptor was activated in a concentration-dependent manner by
two peptides encoded on flp-2, SPREPIRF-NH2 (FLP-2A) and LRGEPLRF-NH2 (FLP-2B) (Fig.
2). The expressed receptor was functional in transfected cells cultured either at 28°C or 37°C
(Fig. 2A). This is exemplified by the responses to increasing concentrations of FLP-2A, with
EC50 values and 95% confidence limits of 18.5 nM (14.7-23.2 nM) and 7.7 nM (6.3-9.5 nM)
under 28°C and 37°C cell culture conditions, respectively. It can be concluded that the FLP-2R
118 receptor was slightly more functional at 37°C than at 28°C (based on non-overlapping 95%
confidence limits).
The FLP-2A and FLP-2B peptides were equally potent in cells cultured at 37°C with EC50 values
and 95% CI of 3.5 nM (1.59 – 7.6) and 6.5 nM (4.2-10.2), respectively (Fig. 2B). The ligandevoked Ca2+ signal was completely abolished in cells pretreated with the phospholipase inhibitor
U-73122 (Fig. 2B), suggesting that the FLP-2 receptor couples to Gαq signaling pathways in
these cells.
Figure 1. Alignment of predicted protein sequences of T19F4.1 and obtained clones. T19F4.1a and b
correspond to the receptor sequences reported in WormPep and Ce50b, c and d correspond to the clone sequences
obtained from cDNA amplification and cloning. The CLC sequence viewer was used to generate this multiple
alignment in which identical residues are represented by dots.
119 Figure 2. Functional expression of FLP-2R/T19F4.1 in CHO cells; Ca2+ mobilization assay by FLIPR. A.
Activation of the receptor by FLP-2A was temperature-independent. B. FLP-2R was equally effectively activated by
FLP-2A and FLP-2B in cells cultivated at 37°C (EC50 values for the peptides were not statistically different, p >
0.05). Pre-incubation with U-73122 prevented Ca2+ release in response to FLP-2A, indicating that FLP-2R is
coupled to the Gαq signaling pathway.
3.3.
FLP-2R functional expression in yeast
Expression of FLP-2R in S. cerevisiae generated a strain in which growth in the absence of
exogenous histidine in the medium was only possible in the presence of a receptor agonist, such
as either of the FLP-2 peptides (Fig. 3). Growth in the presence of these peptides only occurred
in the S. cerevisiae strain that co-expresses the Gαq chimera; no agonist-dependent growth (at the
EC80 concentration for the Gαq chimera strain) was observed in yeast strains expressing FLP-2R
and other Gα chimeras or in yeast transformed with the empty vector, although all these strains
grew normally in the presence of histidine (not shown). In the Gαq chimera strain (CY13397),
FLP-2A had an EC50 value and 95% CI of 71 (66 – 78) nM, with a corresponding value of 127
(121-134) nM for FLP-2B (Fig. 3). None of the other tested peptides had potency within a factor
of 10 of FLP-2A or FLP-2B (not shown), suggesting again that FLP-2R is the most relevant
receptor for them.
120 Figure 3. FLP-2R/T19F4.1 activation by FLP-2 peptides in yeast. The peptides FLP2-A (SPREPIRFa) and
FLP2-B (LRGEPIRFa) activated T19F4.1 in a concentration-dependent manner when the receptor is expressed in S.
cerevisiae strain CY13397 which co-expresses a chimeric Gαq protein. Receptor activation was quantified using an
Alamar Blue fluorescence assay. Data were normalized to the highest value and presented as the mean and SEM of
three experiments (n = 3).
4.
Discussion
Analysis of the C. elegans genome has identified ~ 60 genes encoding predicted GPCRs that are
likely to have peptide ligands (Bargmann, 1998; Keating et al., 2003). Only a subset of these
receptors has been matched to a ligand (see Lowery et al., 2003; McVeigh et al., 2006; Husson et
al., 2007). Among them is T19F4.1, reported as the FLP-2 receptor (Lowery et al., 2003;
Mertens et al., 2005). As part of a large-scale program to match C. elegans and Drosophila
melanogaster putative peptide GPCRs with their ligands for screen development (Larsen et al.,
2001; Lowery et al., 2003; Greenwood et al., 2005; Woods et al., 2010), our group had also
identified this receptor as being the target for peptides encoded on the flp-2 precursor gene
(Lowery et al., 2003). However, in light of some differences in pharmacology apparent in
comparing the data from the two laboratories and the expression of this invertebrate peptide
GPCR in yeast in a format suitable for use in high-throughput screening (Geary, 2012; Geary and
Ubalijoro, 2012), publication of the current data is warranted.
121 The most pronounced difference in the two sets of experiments on expression of FLP-2R in
mammalian cells is the difference in potency observed. In the work of (Mertens et al., 2005),
peptide FLP2-A activated the FLP-2R with EC50 values in the high nanomolar range, while the
FLP2-B peptide was active only at micromolar concentration and did not saturate the receptor
even at 100 µM. In contrast, we found both peptides to be roughly equipotent, with EC50 values
in the range of 5 nM for FLP-2R expressed in mammalian cells (Fig. 2B). These differences in
FLP-2R pharmacology might be due to the use of different cells for expression, which were
CHO-10001A cells in the current studies and CHO-K1 cells in (Mertens et al., 2005). Potency
values in the nM range are more consistent with many other peptide-GPCR systems, including
examples from C. elegans (Kubiak et al., 2003; McVeigh et al., 2006; Kubiak et al., 2007). The
potencies reported in the current work are also more consistent with the physiological effects of
the flp-2 peptides on nematode (Ascaris suum) muscle physiology; FLP-2A and B had
concentration-dependent excitatory effects on dorsal and ventral muscle strips of A. suum. The
activity thresholds for these peptides were 10 nM (LRGEPIRFamide) and 3 nM
(SPREPIRFamide) (Geary, unpublished observations). The flp-2-encoded peptides were also
active in bioassays using the A. suum ovijector (Moffett et al., 2003) and C. elegans pharynx
(Papaioannou et al., 2005); again, responses were observed at concentrations as low as 10 nM in
these studies. That the current data reveal the same level of potency for biological activity of
these peptides and activation of FLP-2R expressed in mammalian cells adds confirmation that
this is likely to be the physiologically relevant pairing.
Based on pharmacological evidence with a phospholipase inhibitor, we conclude that FLP-2R
signals through the Gαq pathway in CHO cells. This association was confirmed in the yeast
system; FLP2-induced growth of recombinant yeast expressing FLP-2R was only observed in
strains that also expressed the Gαq chimeric construct. Conservation of the signaling pathway
suggests that the function of the receptor-G protein interaction is maintained in two vastly
different heterologous systems, but does not prove that this interaction is also operative in situ.
Null mutations in the Gαq locus (EGL-30) are apparently lethal in C. elegans, while reduction-infunction alleles have severe paralytic and other consequences (Bastiani and Mendel, 2006),
implicating function of this G protein in multiple pathways. This subunit mediates the signaling
122 pathway for an excitatory C. elegans FLP, FLP-17A (KSAFVRFamide) (Papaioannou et al.,
2008), but evidence is not yet available to link it with the actions of the FLP-2 peptides in C.
elegans.
Expression of flp-2 in C. elegans has been traced to two neurons involved in amphidial circuits,
additional neurons in the pharyngeal region, a neuron in the ventral ganglion and a motorneuron
that innervates dorsal somatic muscle (Kim and Li, 2004). The tissue localization of the FLP-2R
encoded in the T19F4.1 locus has not been reported in this organism, but would add important
and relevant information to the biology of this neuropeptidergic system. Inferences about the
utility of various proteins as targets for anthelmintic discovery are often based on phenotypic
observations of loss-of-function mutations in the genes encoding the protein or as a result of
RNA interference-mediate reduction in mRNA (McCarter, 2004; Behm et al., 2005; Geary,
2012). In that regard, it has been reported that knockdown of the flp-2 gene in the C. elegans rrf3 strain resulted in embryonic lethality or larval arrest and post-embryonic growth defects
(Mousley et al., 2010). However, RNA interference experiments conducted in an rrf-3 strain on
60 putative GPCRs encoded in the C. elegans genome did not reveal a phenotype for T19F4.1
(Keating et al., 2003). In general, neuropeptide systems seem not to be constantly activated, so
that antagonists (or RNA interference or loss-of-function mutations) are often silent. These kinds
of data, coupled with the potent and profound effects of many FLPs on nematode neuromuscular
function (McVeigh et al., 2005), suggest that standard validation of drug targets by inactivation
is unlikely to support FLP receptors as useful proteins for therapeutic intervention. Instead,
agonists are more likely to be useful for chemotherapy.
The yeast system for functional expression of heterologous GPCRs is well suited to screening for
GPCR agonists (Broach and Thorner, 1996; Pausch, 1997; Ladds et al., 2005; Minic et al., 2005;
Wang et al., 2006; Dowell and Brown, 2009). This system is built around the fact that yeast
express a peptide GPCR for which the physiological ligand is the mating pheromone (Wang et
al., 2006; Dowell and Brown, 2009). The engineered yeast strains have null mutations in the
genes encoding the endogenous GPCR, the endogenous Gα subunit and in elements that serve to
desensitize the signaling cascade. These modifications enable heterologous GPCRs to be
expressed in isolation with co-expression of Gα chimeras that help to establish receptor-G
123 protein coupling (Brown et al., 2000; Wang et al., 2006; Dowell and Brown, 2009). In this
system, receptor activation can be coupled to a reporter gene; we chose to use a HIS3 reporter,
which corrects an auxotrophy. Thus, activation of the nematode GPCR permits growth of the
yeast strain on histidine-deficient medium. This system allowed us to confirm the ligand match
for FLP-2R observed in CHO cells. Although the potency of the peptide ligands was
significantly lower in yeast than in mammalian cells, this may simply reflect the barrier
properties of the yeast cell wall, which restrict diffusion of large, charged molecules such as
peptides to the cell membrane (the location of the GPCR). The lower potency of the FLP-2 in
yeast compared to mammalian cells is consistent with results in other nematode GPCR-peptide
pairs in this recombinant yeast system (unpublished observations).
An attractive feature of the recombinant yeast system is its robustness, supporting the potential
use of such strains for screening chemical libraries for non-peptide FLP receptor ligands as
potential anthelmintics. This feature is particularly valuable for operation in resource-limited
settings (Geary, 2012; Geary and Ubalijoro, 2012). Screening for agonists relies on the detection
of growth, as evidence by a change in color or fluorescence, against a null background. Detection
of growth, as opposed to inhibition of growth, requires minimal statistical analysis; any change
above background is of interest. The system also permits co-incubation of multiple strains of
yeast with different receptors (‘multiplex’ format); growth of any strain induced by an agonist is
not affected by the presence of cells expressing other receptors, and the target of the agonist can
be determined in follow-up assays using the component strains in separate wells. This format
also means that the inoculum size can be imprecise and that the time of incubation can vary; both
factors are important considerations in laboratories which may not be fully equipped and to
which travel may not always be reliable. The ability to grow yeast at ambient temperatures, to
detect changes in Alamar blue color visually, and the robustness of the yeast system led to the
implementation of a screening project for non-peptide ligands of FLP receptors as candidate
anthelmintics (Geary, 2012; Geary and Ubalijoro, 2012). Based on the physiological activity of
FLP-2 peptides and the qualities of the FLP-2R yeast strain, this target has been incorporated in
the multiplex system.
124 Concerns about the conservation of pharmacological characteristics for receptors expressed in
heterologous systems may be relevant. In that regard, it is reassuring that there is experimental
evidence to suggest that the ligand-binding profile of GPCRs is maintained whether the receptor
is expressed in yeast or mammalian cells (Brown et al., 2011). Whether this will be true for
nematode GPCRs remains to be proven.
Acknowledgments
We thank Susan Nulf, Marjorie Zantello, Catherine Burton and John Davis (formerly of
Pharmacia Animal Health) for excellent technical assistance and James Broach (Pennsylvania
State University and Cadus, Inc.) for advice on the use of the yeast strains.
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