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

Abstract

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.

Keywords: C. elegans; Cys-loop receptors; anthelmintic; glutamate-activated chloride channels; patch-clamp.

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.

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.

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 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).

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.

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).