A New Antagonist of Caenorhabditis elegans Glutamate-Activated Chloride Channels With Anthelmintic Activity

Article abstract of DOI:10.3389/fnins.2020.00879

Nematode parasitosis causes significant mortality and morbidity in humans and considerable losses in livestock and domestic animals. The acquisition of resistance to current anthelmintic drugs has prompted the search for new compounds for which the free-living nematode Caenorhabditis elegans has emerged as a valuable platform. We have previously synthetized a small library of oxygenated tricyclic compounds and determined that dibenzo[b,e]oxepin-11(6H)-one (doxepinone) inhibits C. elegans motility. Because doxepinone shows potential anthelmintic activity, we explored its behavioral effects and deciphered its target site and mechanism of action on C. elegans. Doxepinone reduces swimming rate, induces paralysis, and decreases the rate of pharyngeal pumping required for feeding, indicating a marked anthelmintic activity. To identify the main drug targets, we performed an in vivo screening of selected strains carrying mutations in Cys-loop receptors involved in worm locomotion for determining resistance to doxepinone effects. A mutant strain that lacks subunit genes of the invertebrate glutamate-gated chloride channels (GluCl), which are targets of the widely used antiparasitic ivermectin (IVM), is resistant to doxepinone effects. To unravel the molecular mechanism, we measured whole-cell currents from GluClα1/β receptors expressed in mammalian cells. Glutamate elicits macroscopic currents whereas no responses are elicited by doxepinone, indicating that it is not an agonist of GluCls. Preincubation of the cell with doxepinone produces a statistically significant decrease of the decay time constant and net charge of glutamate-elicited currents, indicating that it inhibits GluCls, which contrasts to IVM molecular actions. Thus, we identify doxepinone as an attractive scaffold with promising anthelmintic activity and propose the inhibition of GluCls as a potential anthelmintic mechanism of action.

Full text of DOI:10.3389/fnins.2020.00879

ORIGINAL RESEARCH
published: 19 August 2020
doi: 10.3389/fnins.2020.00879
A New Antagonist of Caenorhabditis
elegans Glutamate-Activated
Chloride Channels With Anthelmintic
Activity
María Julia Castro1,2†, Ornella Turani1†, María Belén Faraoni2, Darío Gerbino2 and
Cecilia Bouzat1
*
1 Departamento de Biología, Bioquímica y Farmacia, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB),
Universidad Nacional del Sur (UNS)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Bahía Blanca,
Argentina, 2 Instituto de Química del Sur (INQUISUR), Universidad Nacional del Sur (UNS)-Consejo Nacional
de Investigaciones Científicas y Técnicas (CONICET), Bahía Blanca, Argentina
Nematode parasitosis causes significant mortality and morbidity in humans and
considerable losses in livestock and domestic animals. The acquisition of resistance
to current anthelmintic drugs has prompted the search for new compounds for which
the free-living nematode Caenorhabditis elegans has emerged as a valuable platform.
We have previously synthetized a small library of oxygenated tricyclic compounds
and determined that dibenzo[b,e]oxepin-11(6H)-one (doxepinone) inhibits C. elegans
motility. Because doxepinone shows potential anthelmintic activity, we explored its
behavioral effects and deciphered its target site and mechanism of action on C. elegans.
Doxepinone reduces swimming rate, induces paralysis, and decreases the rate of
pharyngeal pumping required for feeding, indicating a marked anthelmintic activity. To
identify the main drug targets, we performed an in vivo screening of selected strains
carrying mutations in Cys-loop receptors involved in worm locomotion for determining
resistance to doxepinone effects. A mutant strain that lacks subunit genes of the
invertebrate glutamate-gated chloride channels (GluCl), which are targets of the widely
used antiparasitic ivermectin (IVM), is resistant to doxepinone effects. To unravel the
molecular mechanism, we measured whole-cell currents from GluClα1/β receptors
expressed in mammalian cells. Glutamate elicits macroscopic currents whereas no
responses are elicited by doxepinone, indicating that it is not an agonist of GluCls.
Preincubation of the cell with doxepinone produces a statistically significant decrease of
the decay time constant and net charge of glutamate-elicited currents, indicating that it
inhibits GluCls, which contrasts to IVM molecular actions. Thus, we identify doxepinone
as an attractive scaffold with promising anthelmintic activity and propose the inhibition
of GluCls as a potential anthelmintic mechanism of action.
Edited by:
Cesar Mattei,
Université d’Angers, France
Reviewed by:
Neil S. Millar,
University College London,
United Kingdom
Yoav Paas,
Bar-Ilan University, Israel
*Correspondence:
Cecilia Bouzat
inbouzat@criba.edu.ar
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Neuropharmacology,
a section of the journal
Frontiers in Neuroscience
Received: 13 June 2020
Accepted: 28 July 2020
Published: 19 August 2020
Citation:
Castro MJ, Turani O, Faraoni MB,
Gerbino D and Bouzat C (2020) A
New Antagonist of Caenorhabditis
elegans Glutamate-Activated Chloride
Channels With Anthelmintic Activity.
Front. Neurosci. 14:879.
Keywords: C. elegans, Cys-loop receptors, glutamate-activated chloride channels, patch-clamp, anthelmintic
Abbreviations: ECS, extracellular solution; GluCl, glutamate-gated chloride channel; ICS, intracellular solution; IVM,
ivermectin; L-AChR, levamisole-sensitive acetylcholine receptor; N-AChR, nicotine-sensitive acetylcholine receptor; nAChR,
doi: 10.3389/fnins.2020.00879
nicotinic acetylcholine receptor; NGM, nematode growth medium; τ , current decay time constant.
d
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GluCls as Drug Targets
glc-2 (GluClβ), glc-3 (GluClα4), and glc-4 (Cully et al., 1994,
1996; Vassilatis et al., 1997; Dent et al., 2000; Horoszok et al.,
INTRODUCTION
Nematode parasitosis is an important cause of mortality 2001). Functions associated with GluCls include pharyngeal
and morbidity in humans and affects livestock and domestic pumping, which is required for feeding and maintaining
animals. As many as one-third of the world’s population hydrostatic pressure, and for the regulation of locomotion, and
harbors infections with helminths. Also, nematodes have olfactory and temperature responses (Jones and Sattelle, 2008).
an important negative impact on animal productivity Heterologous expression studies have shown that both GluClα1
worldwide. The reduced anthelmintic drug development and GluClβ subunits form functional homomeric receptors, the
and the ever-increasing resistance of nematodes to the limited first responding to IVM and the latter to glutamate (Vassilatis
number of drugs have become a global concern for veterinary et al., 1997; Li et al., 2002), and that GluClα1/β heteropentamers
and human health.
respond to both IVM and glutamate (Dent et al., 1997; Degani-
Parasitic nematodes are not ideal laboratory animals for Katzav et al., 2016). The X-ray structure of the homomeric
drug screening due to the difficulty for genetic manipulation GluClα has revealed information about the binding sites of the
and the need for infected host animals. C. elegans shares allosteric agonist IVM, the orthosteric agonist L-glutamate and
physiological and pharmacological features with parasitic the open-channel blocker picrotoxin (Hibbs and Gouaux, 2011).
nematodes and it is sensitive to most anthelmintic drugs. The
We have recently synthetized a series of oxygenated tricyclic
major neurotransmitter receptors are similar between C. elegans compounds and determined their anthelmintic activity by
and parasitic species (Angstadt et al., 1989; Holden-Dye and measuring rapid effects on C. elegans. The exposure to
Walker, 2007). Thus, the free-living nematode C. elegans has dibenzo[b,e]oxepin-11(6H)-one (doxepinone) produced a rapid
contributed as a parasitic model to defining mechanisms of concentration-dependent decrease of the thrashing rate (IC50
antiparasitic drug action.
∼300 µM), which is a measure of C. elegans motility
Cys-loop receptors are major targets of anthelmintic drugs and swimming rate (Buckingham and Sattelle, 2009; Scoccia
in parasites and C. elegans (Holden-Dye and Walker, 2006; et al., 2017). Doxepinone is considered a privileged structure,
Beech and Neveu, 2015). They belong to the family of which refers to compounds whose scaffolds commonly consist
pentameric ligand-gated ion channels and play key roles of a rigid ring, including heteroring systems that present
throughout the nervous system in vertebrates and invertebrates. appended residues in well-defined orientations required for
They are involved in physiological processes, including muscle target recognition (Evans et al., 1988). Through appropriate
contraction, and are targets for clinically relevant drugs functional group modifications, these scaffolds can provide
(Wolstenholme, 2011). In vertebrates, Cys-loop receptors include ligands for a number of functionally and structurally discrete
acetylcholine nicotinic (nAChRs) and 5-hydroxytryptamine biological receptors, and have, therefore, attracted interest
type 3 (5-HT3) receptors, which are cationic channels, and across a broad spectrum of sciences from chemistry and
GABAA and glycine receptors, which are anionic channels. biology to medicine. Because doxepinone is a privileged
C. elegans and parasitic nematode muscle contains two different structure with potential anthelmintic activity, we here explored
types of nAChRs, a levamisole-sensitive (L-AChR) and a in detail its behavioral effects and deciphered its target
nicotine-sensitive (N-AChR), and a GABA receptor. L-AChRs site and mechanism of action on C. elegans as a parasite
mediate muscle contraction whereas GABA receptors mediate model. We propose doxepinone as an attractive scaffold
muscle relaxation. These two receptors are essential for the with potential antiparasitic activity mediated, at least in
typical sinusoidal movement and are targets of anthelmintic part, through GluCls.
compounds, like levamisole and pyrantel (L-AChR agonists)
and piperazine (GABA receptor agonist) (Martin, 1997; Fleming
MATERIALS AND METHODS
Synthesis of Doxepinone
et al., 1997; Culetto et al., 2004; Towers et al., 2005;
Bamber et al., 2005; Rayes et al., 2007). N-AChR is a
homopentameric receptor that responds to acetylcholine and
nicotine and its role in locomotion is not fully understood Dibenzo[b,e]oxepin-11(6H)-one (named as doxepinone) was
(Touroutine et al., 2005). Compared to vertebrates, invertebrates synthetized following the protocol developed by our group
contain a larger variety of Cys-loop receptors, including a (Scoccia et al., 2017 and Supplementary Figure S1). Briefly,
unique type of glutamate-gated chloride channels (GluCl) 2-(phenoxymethyl) benzoic acid was prepared by treating the
(Jones and Sattelle, 2008).
commercially available isobenzofuran-1(3H)-one with sodium
GluCls are of considerable medical and economical phenoxide, which was obtained by reacting phenol with NaH in
importance because they are targets of macrocyclic lactones, the presence of DMF at reflux. 2-(phenoxymethyl) benzoic acid
such as ivermectin (IVM), which are the most widely used was then cyclized by intramolecular acylation from the carboxylic
antiparasitic drugs (Chen and Kubo, 2018). IVM is used in acid compound by using FeCl2 and dichloromethyl methyl ether
veterinary for gastrointestinal roundworms, lungworms, grubs, as cooperative system in the presence of dichloromethane at
sucking lice, and mange mites and in humans for treating filarial room temperature. Purity was determined by elemental analysis
diseases (Campbell, 2012).
and melting point.
There are six C. elegans genes encoding GluCl subunits:
L-Glutamic acid monosodium salt hydrate, ivermectin and
avr-14 (GluClα3 subunit), avr-15 (GluClα2), glc-1 (GluClα1), dimethyl sulfoxide were from Sigma-Aldrich Chem Co.
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plates at concentrations lower than 1% v/v. Wild-type and
DA1316 young adult worms were transferred to drug plates and
allowed to remain at 20◦C for a 30 min period. We performed
3 independent whole experiments with different worm batches.
Each experiment included comparison of the effects of drugs on
wild-type and mutant strains in parallel, with n = 14 animals for
each condition in each experiment. The number of contractions
in the terminal bulb of the pharynx (pumps per minute) was
counted using a stereomicroscope at 50× magnification.
Caenorhabditis elegans Strains and
Culture
Nematode strains used were: N2: Bristol wild type; and
the null mutants of Cys-loop receptor subunits: RB918: acr-
16(ok789); DA1316: avr-14(ad1302);avr-15(ad1051);glc-1(pk54);
CB382: unc-49(e382); CB904: unc-38(e264); MT9668: mod-
1(ok103). All strains were obtained from the Caenorhabditis
Genetic Center, supported by the National Institutes of Health
- Office of Research Infrastructure Programs (P40 OD010440).
Nematodes were maintained at 21◦C using standard culture
methods (Brenner, 1974; Stiernagle, 2006; Hernando et al., 2012,
2019). Assays were carried out following standard protocols
described in WormBook1.
Body Length Measurements
Young adult hermaphrodite worms (n = 10 worms for each
condition) were transferred to NGM plates containing 2.5 mM
doxepinone. Vehicle (1% DMSO) was used as a control. After 2 h,
images were acquired with a digital camera ToupCam UCMOS
05100KPA (Toup Tek Photonics) and body length was measured
using FIJI-ImageJ software. Four independent whole experiments
were analyzed in parallel with the controls.
Locomotion and Paralysis Assays
All behavioral assays were done at room temperature (21–23◦C)
and all comparisons were done in parallel. Assays were performed
with young adult hermaphrodite worms from synchronized
plates. For comparison among different drug concentrations
or strains, the assays of the control and different conditions
were performed simultaneously. For each condition, 30 worms
(n = 30) were used in paralysis assays and 20 worms (n = 20) in
thrashes assays. Each condition was evaluated 4 times in different
days with different worm batches and always in parallel with the
control, as described before (Hernando et al., 2012, 2019).
Thrashing assays were performed in 100 µl M9 buffer in the
absence or presence of the drug in a 96-well microliter plate as
described before (Jones et al., 2011). A single thrash was defined
as a change in the direction of bending at the mid body. All assays
were carried out by two independent operators and were blinded
to the sample identities.
Heterologous Cell Expression of GluCls
GluCls were transiently expressed in BOSC 23 cells, which are
modified HEK 293T cells (Pear et al., 1993). The cDNAs encoding
the C. elegans GluClα1 (containing gfp between transmembrane
domains M3 and M4) and GluClβ subunits, both subcloned into
the pcDNA3.1 vector, were kindly provided by Dr. Paas (Degani-
Katzav et al., 2016). Cells were transfected by calcium phosphate
precipitation with the subunit cDNAs (total 4 µg/35 mm dish) at
a ratio GluClα1:GluClβ 1:1 essentially as described before (Bouzat
et al., 2008; Nielsen et al., 2018). All transfections were carried
out for about 8–12 h in DMEM with 10% fetal bovine serum
and were terminated by exchanging the medium. Cells were used
for whole-cell recordings 2 or 3 days after transfection, time at
which maximal functional expression levels are typically achieved
(Bouzat et al., 2008).
Paralysis was determined on agar plates containing the tested
drug at room temperature as described before (Hernando et al.,
2012). Body paralysis was followed by visual inspection at the
indicated time (up to 120 min) and was defined as the lack of
body movement in response to prodding.
Whole-Cell Recordings From BOSC23
For prodding, we used the gentle touch stimulus delivered to
the body with an eyebrow hair, avoiding touching the animals
too near the tip of the head or tail (Hernando et al., 2019). We
evaluated different types of nematode paralysis: flaccid paralysis
in which worms appear lengthened; spastic paralysis in which
worms appear shorter, and stationary paralysis in which worms
are immobile but respond to prodding by contracting body wall
muscle (Kass et al., 1980; Hernando et al., 2019). Videos were
acquired with a digital camera ToupCam UCMOS 05100KPA
(Toup Tek Photonics).
Cells
Macroscopic currents were recorded in the whole-cell
configuration as described previously (Bouzat et al., 2008;
Corradi et al., 2009).
The pipette was filled with intracellular solution (ICS)
containing 134 mM KCl, 5 mM EGTA,1 mM MgCl2, and 10 mM
HEPES (pH 7.3). The extracellular solution (ECS) contained
150 mM NaCl, 0.5 mM CaCl2, and 10 mM HEPES (pH 7.4). After
the whole cell formation, ECS containing the agonist or drug was
rapidly applied using a three-tube perfusion system with elevated
solution reservoirs for gravity-driven flow and switching valves
controlled by a VC3 controller (ALA Scientific). The solution
exchange time was estimated by the open pipette method as
described by Liu and Dilger (1991). This method consists in
applying a pulse of 50% diluted ECS to an open patch pipette,
which produces a sudden change in the current measured by
patch-clamp amplifier. After proper adjustment of the electrode
position, the current jump in our system varied between 0.1 and
1 ms (Corradi et al., 2009; Andersen et al., 2016). The compound
doxepinone was dissolved in ECS from DMSO stock solutions.
Stock solutions of ivermectin (IVM) and doxepinone were
in dimethyl sulfoxide (DMSO). For all assays, the final
concentration of DMSO was lower than 1%.
Pharyngeal Pumping Measurements
Measurements were performed with young adult worms on agar
plates. Plates contained 1 µM IVM, 50 or 100 µM doxepinone.
DMSO was used as the vehicle and was present in all control
1www.wormbook.org
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The final concentration of DMSO used to solubilize doxepinone in liquid medium (Scoccia et al., 2017). After 10 min exposure,
was lower than 0.2%. To study the modulatory action of the compound produced a concentration-dependent decrease of
doxepinone, responses were evaluated following co-application the thrashing rate (IC50 ∼300 µM) (Scoccia et al., 2017). Because
or preincubation protocols. After whole cell formation, 3 mM doxepinone is an interesting synthetic molecule with potential
glutamate-elicited currents (control currents) were first recorded anthelmintic activity we sought to explore its anthelmintic effects
by a pulse (6 s) of ECS containing glutamate. The compound and identify target sites.
was then co-applied with glutamate (Co-application protocol)
To determine receptor targets involved in the rapid effects of
or applied during 1 min in the absence of glutamate before doxepinone on C. elegans swimming rate, we explored the effects
the second glutamate pulse (Preincubation protocol). For all on selected mutant strains lacking Cys-loop receptors involved
experiments, the duration of the glutamate pulse was 6 s and in worm locomotion, which are targets of anthelmintic drugs.
the time of recording was 8 s. A 60-s wash with ECS alone The screening is based on the hypothesis that the absence of the
allowed total recovery of control currents. The treated currents target site leads to drug resistance and, therefore, in the mutant
were normalized to currents elicited by glutamate alone in the worm, the thrashing rate in the presence of doxepinone will
same cell (control current). At the end of the protocol, the not be affected. Since some mutant strains show uncoordinated
control current was again tested and the experiments in which the phenotypes, we determined the number of thrashes/min of
currents were reduced to more than 80% of the original control wild-type and each mutant strain in liquid medium in the
current were discarded.
absence and presence of doxepinone (Figure 1A). For each
Currents were filtered at 5 kHz and digitized at 20 kHz strain, 20 worms in each condition were used, and experiments
using an Axopatch 200B patch-clamp amplifier (Molecular were repeated with different worm batches and in different
Devices, CA, United States) and acquired using WinWCP days in four independent assays, always in parallel with the
software (Strathclyde Electrophysiology Software, University of control condition.
Strathclyde, Glasgow, United Kingdom). The recordings were
The thrashing rate of wild-type worms in M9 buffer (plus 1%
analyzed using the ClampFit software (Molecular Devices, CA, DMSO) was 204 ± 9.3 min−1 (Figure 1A). After 30 min pre-
United States). Currents were fitted by a single exponential exposure to 0.1 mM doxepinone, this rate decreased about 50%
function according to the equation:
(103 thrashes/min, p < 0.001, Student t-Test) (Figure 1A).
Mutant worms lacking the UNC-38 subunit (CB904 strain),
which is an essential L-AChR subunit, or lacking UNC-49
(GABA) receptors (CB382 strain) showed lower thrashing rates
than wild-type worms as well as uncoordinated phenotypes
(Brenner, 1974, Figure 1A). Nevertheless, doxepinone reduced
the trashing rate in both mutants (Figure 1A, p < 0.001), with
the magnitude of the reduction being similar to that observed
in wild-type worms (Figure 1B). The MT9668 strain lacks the
serotonin-gated chloride channel, MOD-1, that is involved in
locomotion and behavior (Ranganathan et al., 2000; Komuniecki
et al., 2012). This mutant strain was sensitive to the drug
(Figure 1A, p < 0.001), which produced a decrease of the trashing
rate similar to that observed in wild-type worms (Figure 1B).
Thus, L-AChR, UNC-49 and MOD-1 receptors are not the main
receptors involved in the rapid effects of doxepinone in the
swimming rate. The RB918 strain, which lacks the nicotine-
sensitive nAChR (N-AChR) present in muscle (Touroutine
et al., 2005), was also sensitive to doxepinone (Figure 1A,
n = 20 worms for each condition). However, the decrease
of the trashing rate was slightly, but statistically significantly,
lower than that observed for wild-type worms, indicating some
type of contribution of this receptor to doxepinone action
(Figure 1B, p < 0.01).
I(t) = I[exp(-t/τ )] + I
∞
d
in which t is time, I is the peak current, I is the steady state
∞
current value, and τ is the decay time constant. Net charge was
d
calculated by current integration (Andersen et al., 2016). The rise
time (tr10−90%) corresponds to the time taken by the current to
increase from 10 to 90% of its maximal value.
Data and Statistical Analysis
Experimental data are shown as mean ± SD. Statistical
comparisons were done using two-tailed Student’s t-test
for pairwise comparisons or oneway ANOVA followed by
Bonferroni’s post hoc tests for multiple comparisons. All the
tests were performed with SigmaPlot 12.0 (Systat Software,
Inc.). Statistically significance was established at p-values < 0.05
(∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Concentration–response
curves were determined by non-linear regression fits to
the Hill equation using Prism 5.0 (GraphPad, San Diego,
CA, United States).
RESULTS
The thrashing rate of the triple mutant worms lacking three
GluCl subunit genes (DA1316) was similar to that of wild-type
animals in the absence of the drug (Figure 1A). Interestingly,
exposure to 0.1 mM doxepinone did not affect this rate, in
contrast to the effects observed in wild-type worms (Figure 1A,
n = 20, p > 0.05). The percentage of the reduction of the
thrashing rate due to the presence of doxepinone was statistically
1-Screening of C. elegans Mutant Strains
for Resistance to
Dibenzo[b,e]oxepin-11(6H)-One
(Doxepinone): Measurements of
Swimming Rates
We have previously found that dibenzo[b,e]oxepin-11(6H)-one, significantly different between wild-type and DA1316 worms
referred to as doxepinone, produced rapid paralysis of C. elegans (Figure 1B, p < 0.001). Altogether, our results indicate that
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GluCls are involved in the rapid effect of doxepinone on
the swimming rate.
O
O
2-Doxepinone-Induced Paralysis Assays
dibenzo[b,e]oxepin-11(6H)-one (doxepinone)
on Agar Plates
To determine the type of paralysis and the contribution of the
different Cys-loop receptors to doxepinone effects, we performed
paralysis assays on agar plates containing the drug.
A
250
***
***
***
ns
We first characterized the type of paralysis exerted by
doxepinone by exposing wild-type worms to 2.5 mM doxepinone
for 30–120 min in agar plates. After 60 min exposure, the worms
in the presence of doxepinone were immobile but respond to
prodding by contracting body muscle (Supplementary Video
S1). After 2 h exposure, worms did not show any response and
were completely paralyzed. The length of worms in the absence
of the drug was 1.16 ± 0.01 mm whereas it was 1.10 ± 0.04 mm
after 1 h exposure (n = 10, p > 0.05), indicating neither spastic
nor flaccid paralysis.
200
150
100
50
***
***
For wild-type worms, a clear concentration- and time-
dependent paralysis was detected during the 2 h assay at
a 1–3 mM doxepinone concentration range (Figure 2A). At
the maximum exposure time (2 h), ∼85% adult worms were
paralyzed by 3 mM doxepinone (Figure 2A, n = 30). The IC50
values determined for the inhibition of moving worms in agar
plates were 2.58 ± 0.01 mM and 2.10 ± 0.01 mM for 90 and
120 min exposure, respectively (Figure 2B). Paralysis assays on
agar plates measured at short times usually require higher drug
concentrations than those used in liquid medium probably since
the drug must be absorbed from the solid phase.
To confirm the contribution of the different Cys-loop
receptors to doxepinone effects, we next measured paralysis
as a function of time of different mutant worms exposed
to 2.5 mM doxepinone. We found that the reduction of
moving worms as a function of time of exposure for MT9668,
CB904, and CB382 strains did not differ from that of wild-
type worms (Figure 2C). For these mutants, the fraction of
moving worms was reduced to ∼0.40 after 2 h exposure
on agar plates containing 2.5 mM doxepinone (Figure 2C).
Although slight, there was a statistically significant difference
in the effects of the drug on RB918 worms (lacking N-AChR)
respect to wild-type worms after 2 h exposure (p < 0.01,
n = 30). For worms lacking GluCl subunits (DA1316), only a
slight reduction of the fraction of moving animals was detected
after 2 h exposure to doxepinone (18%). This reduction was
markedly different to that of wild-type worms and other mutants
(∼60%), again indicating that GluCls are involved in doxepinone
paralysis (Figure 2C).
0
doxepinone
-
+
- +
CB904 CB382
L-AChR GABAR
- +
-
+
- + - +
RB918 DA1316
N-AChR GluCl
MT9668
Worm strain WT
Receptor
MOD-1
B
1.2
1.0
0.8
0.6
0.4
0.2
0.0
***
**
ns
ns
ns
Worm strain
Receptor
RB918 DA1316
N-AChR GluCl
WT
CB904 CB382 MT9668
L-AChR GABAR
MOD-1
FIGURE 1 | Screening of mutants for resistance to doxepinone. Synchronized
young adult wild-type worms were used. Measurements were performed in
liquid medium after 30 min incubation in M9 buffer containing 1% DMSO in
the absence or presence of 0.1 mM doxepinone. A single thrash was defined
as a change in the direction of bending at the mid body. The non-functional
receptor in each mutant strain is indicated. n = 20 worms per condition,
repeated in 4 different days and worm batches, in parallel with the control.
(A) Bar chart showing the thrashing rate for each mutant in the absence (left
bar) or presence of doxepinone (right bar) for each mutant. Statistical
comparisons were made for each strain in the absence and presence of
doxepinone. (B) Bar chart showing the reduction in the thrashes/min in each
strain due to the presence of doxepinone. ns, non-statistically significant,
**p < 0.01 or ***p < 0.001 respect to the change in the wild-type strain.
Because GluCls are targets of IVM we compared the effects of
doxepinone with those of IVM on DA1316 (lacking three GluCl
genes) and wild-type strains. After 30 min exposure to 10 µM
IVM in liquid medium, wild-type worms were fully paralyzed
whereas mutant worms (DA1316) showed only 50% reduction
of the thrashing rate (Figure 3A). In agar plates containing 300
µM IVM, wild-type animals showed a time-dependent paralysis
that yielded 80% paralyzed worms after 2 h. On the contrary,
the percentage of paralyzed mutant worms was smaller than
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GluCls as Drug Targets
A
A
250
200
150
100
50
1.0
0.8
0.6
0.4
0.2
0.0
1.0 mM
1.5 mM
2.0 mM
2.5 mM
3.0 mM
***
0
20
40
60
80 100 120
Time (minutes)
B
1.0
0.8
0.6
0.4
0.2
0.0
90 min
120 min
***
0
IVM
-
+
-
+
IC50=2.58 mM
IC50=2.10 mM
Worm strain
WT
DA1316
B
100
80
60
40
20
0
-3.4
-3.1
WT
-2.8
-2.5
-2.2
Log [doxepinone] M
C
1.0
***
**
0.8
0.6
0.4
0.2
0.0
WT
DA1316
CB904
CB382
MT9668
RB918
DA1316
0
20
40
60
80
100
120
0
20
40
60
80
100 120
Time (minutes)
Time (minutes)
FIGURE 3 | Effects of ivermectin on wild-type and mutant worms lacking
GluCl genes. Synchronized young adult worms from wild-type worms or
DA1316 strain that lacks GluCl subunit genes. (A) Measurements were
performed in liquid medium after 30 min incubation in M9 buffer plus M9 buffer
containing 1% DMSO in the absence (left bar for each strain) or presence of
10 µM IVM (right bar for each strain). n = 20 worms per condition. The results
correspond to 4 independent assays of the whole experiment shown in the
figure. (B) Percentage of moving worms measured on agar plates as a
function of time of exposure to 300 µM IVM. Statistical comparisons are made
respect to the fraction of wild-type worms. n = 20 worms per condition. The
results correspond to 4 independent assays of the whole experiment shown in
the figure. ***p < 0.001 respect to the change in the wild-type strain.
FIGURE 2 | Fraction of moving animals after exposure to doxepinone as a
function of time and concentration. Synchronized young adult wild-type
worms were placed on agar plates containing DMSO or doxepinone and
observed at the indicated time to determine the fraction of worms that
respond to prodding, which were considered as “moving worms.” The results
correspond to 4 independent assays for all conditions in the figure with 30
worms each time per condition. (A) Wild-type worms were exposed in agar
plates containing doxepinone (1–3 mM range). The fraction of moving animals
was determined at 30 min intervals. (B) Dose-response curves determined on
agar plates for wild-type worms exposed 90 min (gray) or 120 min (black) to
2.5 mM doxepinone. (C) Paralysis as a function of time of exposure of worms
to 2.5 mM doxepinone. Statistical comparisons are made respect to wild-type
strain. **p < 0.01, ***p < 0.001 respect to the change in the wild-type strain.
determined that the type of paralysis of wild-type worms exposed
to 300 µM IVM in agar plates during 60 min was similar to that
10% after 2 h (Figure 3B). After 2 h exposure, the difference observed for doxepinone: worms appeared immobile but respond
in the fraction of moving worms between wild-type and mutant to prodding by contracting body muscle (Hernando and Bouzat,
animals was statistically significative (p < 0.001, n = 30). We also 2014; Supplementary Video S2).
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GluCls as Drug Targets
the actions of doxepinone correlate with those of IVM and
depend on GluCls.
250
200
150
100
50
###
###
###
4-Molecular Actions of Doxepinone on
GluCls
***
To confirm that doxepinone acts at GluCls and to unravel
the mechanism by which it may affect these receptors, we
expressed GluClα1/β in BOSC 23 cells and measured whole-
cell currents at −60 mV holding potential. Currents were
elicited by rapid application of 3 mM glutamate, which is a
concentration higher than its EC50 for these receptors (∼1.5 mM)
but lower than that required for saturation (Cully et al., 1994;
Degani-Katzav et al., 2016).
***
***
***
***
***
0
A
6-s pulse of 3 mM glutamate in ECS elicited
-
0
0
vehicle
-
+
0
0
+
1
0
+
0
50
+
0
+
0
0
+
1
0
+
0
50
+
0
macroscopic currents that reached the peak with a rise
time of 49.30 ± 16.20 ms (n = 9) and decayed in the
presence of the agonist due to desensitization. Typically,
peak currents varied between 2000 and 7000 pA. The decay
was fitted by a single component with a time constant of
2100 ± 1040 ms (n = 9). The application of another pulse of
glutamate after a 20-s wash with ECS allowed full recovery
of the peak current, indicating that most receptors recovered
from desensitization in the absence of the agonist after this
period (Figure 5A).
IVM
doxepinone
0
0
100
100
WT
DA1316
FIGURE 4 | Ivermectin and doxepinone effects on pharyngeal pumping.
Wild-type and DA1316 young adult worms were transferred to
bacteria-seeded NGM plates containing different concentration of IVM and
doxepinone (IVM = 1.0 µM, doxepinone = 50 and 100 µM). After 30 min of
drug exposure, the number of contractions in the terminal bulb of the pharynx
(pumps) was counted using a stereomicroscope at 50x magnification. Bars
represent the mean ± SD from n = 14 animals per condition. Statistical
differences compared to the non-treated condition of the same strain
(***p < 0.001). The symbol # indicates statistically significant differences
between the two strains at the same condition (###p < 0.001).
To further characterize GluCl-mediated currents, we
constructed current-voltage relationships by measuring the
peak current elicited by 3 mM glutamate as a function of the
holding potential (Figure 5B). As shown in the figure, the
magnitude of GluCl-elicited currents increased linearly with the
voltage, indicating an ohmic behavior, and currents did not show
important rectification (n = 4).
3-Doxepinone Reduces Pharyngeal
Pumping Rate
One of the hallmark effects of IVM involving GluCls is
We next proceeded to decipher the molecular actions
pharyngeal pumping inhibition (Dent et al., 1997, 2000). of doxepinone at GluCls. To first determine if doxepinone
We therefore evaluated the effects of doxepinone on can activate GluCls, a 6-s pulse of 0.5 mM doxepinone
the pharyngeal pumping rate of wild-type and mutant (n = 7) or 1 mM doxepinone (n = 3) was applied to cells
worms lacking GluCl subunit genes (DA1316). In the in the whole-cell configuration at -60 mV. No currents
absence of drugs, wild-type and DA1316 worms showed were elicited by doxepinone and sequential application
similar pumping rates of ∼180–200 min−1 (Figure 4). of 3 mM glutamate to the same cell elicited macroscopic
It is important to note that despite lacking GluCls the responses, indicating the presence of functional GluCls
mutant worms show normal pumping due to compensatory (Figure 5C). Thus, doxepinone does not act as an
effects and that IVM also affects other Cys-loop receptors agonist of GluCls.
(Pemberton et al., 2001; Lynagh and Lynch, 2012;
Trojanowski et al., 2016).
We next evaluated the action of doxepinone as
a
modulator of glutamate-activated currents. To this end,
The exposure of worms to 1 µM IVM decreased 4-fold we used two different drug application protocols, one
the pumping rate in wild-type worms and about 1.7-fold in including preincubation of the drug before glutamate
DA1316 worms (n = 14, p < 0.001) (Figure 4). These results application (Preincubation protocol) and the other, application
confirmed that worms lacking GluCl subunits are more resistant of doxepinone together with glutamate (Co-application
to the pumping rate inhibition by IVM than wild-type animals protocol).
(Figure 4). In wild-type animals, increasing concentrations
For the preincubation protocol, a pulse of ECS containing
of doxepinone produced a monotonically decrease of the 3 mM glutamate (6 s-pulse, control current) was first applied
pharyngeal pumping rate, which was reduced 4-fold at 50 to the cell held at -60 mV, and the cell was incubated during
µM and 100% at 100 µM (Figure 4). On the contrary, only 1 min with ECS containing doxepinone (1 mM) before a second
∼1.5-fold reduction was observed in the DA1316 worms in pulse of ECS-glutamate was applied (treated) (Figure 6A). The
the presence of 100 µM doxepinone with respect to the same protocol was repeated three times in the same cell, each
control, thus indicating that the mutants are resistant to the time separated by a 60-s period. An illustrative example of
pharyngeal pumping effect of doxepinone (Figure 4). Thus, an experiment from a single cell is shown in Figures 6A,B.
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August 2020 | Volume 14 | Article 879

Castro et al.
GluCls as Drug Targets
A
C
ECS (20 s)
1 mM doxepinone
3 mM glutamate (6 s)
3 mM glutamate (6 s)
1000 pA
1 s
500 pA
1 s
3 mM glutamate
B
100
60
1
I/Imax
20
20
-20
-60
-100
0.5
3 mM glutamate
Vm
100
-100 -60 -20
60
-0.5
-1
500 pA
1 s
FIGURE 5 | Macroscopic responses of GluClα1/β receptors heterologously expressed in mammalian cells. (A) Macroscopic currents elicited by 3 mM glutamate in
the whole-cell configuration. Representative responses to glutamate in a single cell. Cells were first exposed to a 6-s pulse of 3 mM glutamate, then to a 20-s pulse
of ECS alone, and finally again to the 3 mM glutamate-containing ECS. Pipette potential: -60 mV. (B) Left: currents elicited by 6 s -pulse of 3 mM glutamate at
different pipette potentials (from 100 to -100 mV, Vm). Right: Current (I)-Voltage (Vm) relationship for GluCl channels under symmetrical chloride solutions (n = 4). (C)
Cells were first exposed to a 6-s pulse of 3 mM glutamate (black line current), then to a 6-s pulse of 1 mM doxepinone (violet line current), and finally again to the
3 mM glutamate-containing ECS to verify current recovery. Pipette potential: −60 mV.
The results from different cells were averaged and shown in of doxepinone inhibited glutamate-activated currents but
Figure 6C.
the changes were smaller than those determined under the
Preincubation of the cell with 1 mM doxepinone produced preincubation protocol.
a slight decrease of the peak current and a profound decrease
of the current decay time constant and net charge (Figure 6C).
Compared to the corresponding control current in each cell,
the peak current was reduced to 0.81 ± 0.08 (p = 0.001), the
DISCUSSION
decay rate to 0.57 ± 0.13 (p = 0.001), and the net charge We here identified dibenzo[b,e]oxepin-11(6H)-one (doxepinone)
to 0.49 ± 0.11 (p = 0.002, n = 6). Thus, the main effect of as a novel anthelmintic compound acting through GluCls by
the drug on glutamate-elicited currents is the increase in the a different mechanism to that of the widely used anthelmintic
decay rate (or decrease of the decay time constant), which drug, IVM, and proposed that GluCl inhibition may be further
leads to a concomitant decrease in the net charge. We also explored as a mechanism of action of anthelmintic drugs.
determined that the percentage of changes after drug application
The anthelmintic action of doxepinone is revealed by
were similar among the three applications on each cell. Also, no the induction of worm paralysis measured in agar plates,
significant differences were found in the relative peak current the inhibition of worm mobility in liquid medium, and an
(1.01 ± 0.10), net charge (0.99 ± 0.17), and decay time constant important decrease of the pumping rate. Because rapid effects
(0.95 ± 0.06) with respect to the control when preincubation was as the ones observed for doxepinone may be mediated by
performed with 0.2% DMSO in ECS in the absence of doxepinone ion channels, we performed the first screening for resistance
(n = 4).
to doxepinone on selected null mutant strains lacking Cys-
We also explored if doxepinone applied together with loop receptors involved in worm locomotion. The screening
glutamate (co-application protocol) affected GluCl currents identified GluCls, which are main receptor targets of IVM, as
(Figures 6E,D). In each cell, we applied three pulses of targets of doxepinone. In close agreement with this finding,
glutamate containing ECS, each one separated by 20 s, we demonstrated that the effects of doxepinone recapitulate
and then three pulses of ECS containing 3 mM glutamate those of IVM. Particularly, the inhibition of the pumping rate
and
1 mM doxepinone (Figure 6D). Doxepinone did is a hallmark of IVM action. Also, the paralysis induced by
not produce statistically significant changes in the peak doxepinone is neither spastic nor flaccid as that observed in the
currents (0.99 ± 0.02, p = 0.319) but produced a slight presence of IVM.
and statistically significantly decrease of the net charge
Our screening showed that N-AChRs may play a role in the
(0.83 ± 0.06, p = 0.001) and decay time constant (0.76 ± 0.10, paralysis caused by doxepinone since the worms lacking the ACR-
p = 0.008) (n = 5 cells) (Figure 6E). Thus, co-application 16 subunit are less sensitive to the drug than wild-type worms.
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GluCls as Drug Targets
Preincubation +/-
A
C
1.0
0.8
0.6
0.4
0.2
0
glutamate
glutamate
doxepinone
glutamate
wash
**
***
**
500 pA
2 s
Net charge
B
Peak
d
1.0
0.8
0.6
0.4
0.2
0
c
t
c
t
c
t
Peak
Net charge
d
E
1.0
0.8
0.6
0.4
0.2
0
***
**
c t1 w1 t2 w2 t3
c t1 w1 t2 w2 t3
c t1 w1 t2 w2 t3
D
Co-application -/+
glutamate
glutamate/doxepinone
c
t
c
t
c
t
2000 pA
1 s
Net charge
Peak
d
FIGURE 6 | Effects of doxepinone on glutamate-elicited responses using different application protocols. Doxepinone (1 mM) was applied before the 3 mM glutamate
pulse (preincubation, ± protocol) or together with glutamate (co-application, −/ + protocol). Pipette potential: −60 mV. (A) ± protocol: 6 s-pulse of 3 mM glutamate
was applied before (control current, c) and after (treated current, t) preincubation during 60 s with 1 mM doxepinone. Currents were recorded after 1 min wash with
ECS to confirm recovery (recovered, w). This protocol was repeated 3 times in each cell. (B) Illustrative example of the changes in the macroscopic current
parameters obtained after each application of 3 mM glutamate in a single cell with the protocol shown in (A). c, control current; t and w, correspond to the
glutamate-activated current obtained after 60-s incubation with 1 mM doxepinone (treated, t) or buffer alone (wash, w). The sub index corresponds to the
agonist-application order in the series. (C) Bar chart showing the averaged changes in peak current, decay time constant and net charge due to the preincubation
with 1 mM doxepinone (t). For each experiment, the peak current, the decay time constant and total area were related to those of the control current in each cell (c).
The values correspond to the mean of 6 different cells and 4 different days of transfection (***p < 0.001; **p < 0.01). (D) −/ + protocol: 6 s-pulse of 3 mM glutamate
was first applied alone (control current, black line) and then together with 1 mM doxepinone (treated current, violet line). (E) Bar chart showing the effects of 3 mM
glutamate/1 mM doxepinone co-application on peak current, decay time constant and net charge. The values correspond to the mean ± SD of 5 different cells
(***p < 0.001; **p < 0.01; *p < 0.05).
However, the effect is not as relevant as that mediated by GluCls.
In C. elegans, the expression pattern of the different GluCl
Also, we cannot discard that other receptors, not explored here, genes has been explored (Holden-Dye and Walker, 2014). In
may be involved in doxepinone effects on C. elegans. Finally, particular, GluClα1 subunit expresses in body wall muscle, head
it would be also interesting to perform studies on parasitic neurons, and pharyngeal muscle cells and GluClβ subunit in
nematodes to confirm that they respond to doxepinone similarly pharyngeal muscle pm4 cells. Although GluCl subunits can form
to the free-living nematode.
homomeric or heteromeric receptors in heterologous expression
Inhibitory GluCls are expressed on neurons and muscle systems, the composition of the native receptors remains mostly
across protostome phyla, including mollusks, flatworms, unknown. To elucidate the molecular functional consequences of
nematodes, ticks, and mites, as well as insects and crustaceans doxepinone acting at GluCls, we used as a model the GluClα1/β
(Wolstenholme, 2012). They are of great importance since receptor, which has been previously characterized in detail in
they are one of the main target sites for parasitic control. mammalian cells (Degani-Katzav et al., 2016). Since the DA1316
IVM and macrocyclic lactones are used to eliminate strain lacks three GluCl genes, avr-14, avr-15, and glc-1, it would
nematode infections from millions of humans suffering be interesting to explore in future work the molecular effects of
from diseases, such as river blindness and lymphatic filariasis, doxepinone on other GluCl subtypes (Brockie and Maricq, 2006).
and are also used in domestic pets and cattle for parasitic
Cells expressing GluClα1/β receptors displayed robust
nematodes and ectoparasites (Omura, 2008). GluCls in responses to 3 mM glutamate, in line with previous findings
Anopheles gambiae sensu stricto were also proposed as (Degani-Katzav et al., 2016). By analyzing the peak current as a
targets of IVM to control malaria (Meyers et al., 2015; function of voltage we determined that the ion channel has an
Atif et al., 2019).
ohmic behavior with no significant rectification.
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GluCls as Drug Targets
Currents could not be elicited by the solely application of to that of doxepinone (Scoccia et al., 2017). Interesting, it has
doxepinone, indicating that it is not an agonist of this type been reported that doxepin shows anthelmintic activity against
of GluCl, an action distinct from that exerted by IVM. Pre- the intestinal helminth Ancylostoma ceylanicum, revealing an
exposure of GluCls to doxepinone before activation with the antiparasitic action (Keiser et al., 2016).
agonist significantly decreased the net charge, thus revealing
The widely use of IVM has resulted in selection of resistant
that doxepinone acts as an inhibitor. The analysis of current parasitic nematodes, which has turned into a major global
parameters showed a slight decrease of the peak current and a problem (Laing et al., 2017). It has also raised concerns
significant increase of the current decay rate, indicating that this that IVM resistance may evolve in human parasites as well
latter change governs the negative modulation. The changes are (Laing et al., 2017). Mechanisms underlying IVM resistance
qualitatively similar but quantitatively smaller when doxepinone include changes in sequence and composition of GluCls and in
is applied together with glutamate with no preincubation. proteins regulating membrane permeability and gap junctions.
Receptor inhibition may be caused by competitive or non- Importantly associated to IVM resistance is the enhanced
competitive antagonism. If inhibition were due to competitive expression of the multidrug transport protein, P-glycoprotein,
antagonism, a reduction of the peak current instead of an increase involved in drug exclusion (Blackhall et al., 1998; Xu et al.,
in the decay rate would be observed. Also, the effects produced 1998; Le Jambre et al., 1999; Sangster et al., 1999; Ardelli
by co-application of doxepinone with glutamate would be greater and Prichard, 2013; Ménez et al., 2016). Since IVM and
than those observed with preincubation, which is opposite to our doxepinone are structurally different compounds, they will
experimental results. Thus, our first molecular characterization probably show different activities at P-glycoprotein as well as
suggests that doxepinone acts as a negative allosteric modulator different sensitivities among GluCl subtypes. Thus, our finding
(non-competitive antagonist) of GluCl. The enhancement of the offers a new scaffold for developing new GluCl-active compounds
current decay rate due to the presence of allosteric inhibitors directed to overcome IVM resistance as well as to cover different
may arise from enhanced desensitization or channel block helminth species.
(Gumilar et al., 2003). However, further electrophysiological
The effect of IVM and macrocyclic lactones has been proposed
characterization, including competition studies, is required to to be mediated by increased hyperpolarization due to its agonistic
unequivocally define its mechanism of action as well as its activity at chloride permeable GluCls. We here found that
potential binding sites at GluCl.
doxepinone has the opposite effect. Therefore, the inhibition of
The three-dimensional atomic structure of the homomeric GluCls emerges as an anthelmintic mechanism of action. In line
C. elegans GluClα shows that IVM occupies a cavity between with this, for several insecticides, such as picrotoxin, lindane, and
adjacent subunits in the transmembrane domain (Hibbs and fipronil, the inhibition through GluCl has been proposed as a
Gouaux, 2011). Further studies combining mutant receptors may mechanism involved in their insecticide actions (Narahashi et al.,
help to define if doxepinone binds to the IVM site.
2010; Atif et al., 2019). Fipronil has been shown to reversibly
Although sharing the target receptor, IVM and doxepinone inhibit GluCls from C. elegans (Horoszok et al., 2001) and from
are non-related, structurally different compounds. Whereas IVM Haemonchus contortus (McCavera et al., 2009) and it has been
is a high molecular weight macrocyclic lactone derived from shown to be effective for controlling nematodes on wheat (Cui
avermectins, doxepinone constitutes a class of fused tricyclic et al., 2017). Thus, it appears that both enhanced and reduced
heterocycles present in a variety of bioactive compounds. hyperpolarization can affect worm locomotion and pharyngeal
Structurally, it is typified by the presence of a dibenzo-4- pumping. How neuron wiring underlying the behavioral effects
oxepanone ring system. Each ring is connected in a fused is affected as a result of reduced hyperpolarization due to
formation that does not allow rotation around the carbon- GluCl inhibition is therefore an essential question for future
carbon bonds. This unique structure together with the type studies. Overall, we propose doxepinone as a new scaffold
and position of the linked chemical groups define the with potential antiparasitic activity and the inhibition of GluCls
specific functionalities of doxepinone. The hydrophobic planar as a mechanism of anthelmintic drug action valuable to be
architecture of this oxygenated heterocyclic gives it a remarkably further explored.
different physicochemical behavior with respect to IVM.
The structure of doxepinone is related to that of
tricyclic antidepressants, in particular to doxepin. Tricyclic
antidepressants have been shown to inhibit other Cys-loop
DATA AVAILABILITY STATEMENT
receptors, including vertebrate nAChR and 5-HT3A receptors All datasets generated for this study are included in the
(Gumilar et al., 2003; Gumilar and Bouzat, 2008). Doxepin article/Supplementary Material.
reduced the peak current and increased the decay rate of mouse
muscle nAChRs; these effects were greater when applied during
preincubation than co-applied with the agonist (Gumilar et al.,
2003). Enhancement of desensitization and/or slow channel
AUTHOR CONTRIBUTIONS
blockade was proposed as the mechanism underlying the MC, OT, MF, DG, and CB contributed to study design. MC
macroscopic effects (Gumilar et al., 2003). Although we have and OT acquisition of data. MC, OT, DG, and CB analysis and
shown previously that doxepin slightly decreased the thrashing interpretation of data and contributed to writing. All authors
rate in C. elegans, the effect was significantly lower compared contributed to the article and approved the submitted version.
Frontiers in Neuroscience | www.frontiersin.org
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August 2020 | Volume 14 | Article 879

Castro et al.
GluCls as Drug Targets
FUNDING
SUPPLEMENTARY MATERIAL
This work was supported by the grants from Universidad The Supplementary Material for this article can be found
Nacional del Sur (PGI 24/B227 to CB and PGI 24/Q101 to online at: https://www.frontiersin.org/articles/10.3389/fnins.
DG) and from Agencia Nacional de Promoción Científica y 2020.00879/full#supplementary-material
Tecnológica (PICT-2015-0941 and PICT-2017-1170 to CB and
FIGURE S1 | Total synthesis of doxepinone. Conditions: (A) NaH (1.5 equiv), DMF
PICT-2018-2197 to DG).
(dimethylformamide), reflux, 24 h; then conc. HCl; (B) FeCl2 (0.6 equiv), DCME
(dichloromethyl methyl ether) (1 equiv), DCM (dichloromethane) (0.1 M), rt. Isolated
yield (%) after purification. The details of the procedure have been described
in Scoccia et al. (2017).
ACKNOWLEDGMENTS
VIDEO S1 | Wild-type worms exposed to 2.5 mM doxepinone in agar plates. The
worms are immobile but respond to prodding by contracting body muscles.
We thank the Caenorhabditis Genetics Center and WormBase.
We were grateful to Dr. Paas (Bar-Ilan University, Israel) for
VIDEO S2 | Wild-type worms exposed to 300 µM IVM in agar plates. The worms
are immobile but respond to prodding by contracting body muscles.
generously providing the GluCl subunits.
Cui, J. K., Huang, W. K., Peng, H., Lv, Y., Kong, L. A., Li, H. X., et al. (2017). Efficacy
evaluation of seed-coating compounds against cereal cyst nematodes and root
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Conflict of Interest: The authors declare that the research was conducted in the
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