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Parasit Vectors
2017 Nov 01;101:530. doi: 10.1186/s13071-017-2470-4.
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The novel isoxazoline ectoparasiticide lotilaner (Credelio™): a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls).
Rufener L
,
Danelli V
,
Bertrand D
,
Sager H
.
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BACKGROUND: The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties.
METHODS: In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets.
RESULTS: In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABAA receptor was observed up to a concentration of 10 μM.
CONCLUSIONS: Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate's γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.
Fig. 1. Chemical structures of chloride channel antagonists. The star indicates the chiral center for both isoxazoline molecules. The biologically active compound lotilaner is the S-enantiomer while the R-enantiomer (AHC-2229544) is inactive. Fluralaner is a racemic mixture containing both enantiomers
Fig. 2. Schematic representation of a ligand-gated ion channel subunit. Location of the residues mutated in the transmembrane domains M2 and M3 that have shown to confer resistance to dieldrin and fipronil are represented by a yellow and blue circle respectively. Motifs typical for Cys-loop LGIC such as a large N-terminal extracellular domain, four transmembrane domains (M1-M4), an intracellular loop connecting the M3 and M4 segments and a Cys-loop (2 cysteines) are visible. The aligned amino acid sequences from different species (Dm, Drosophila melanogaster; Cf, Ctenocephalides felis; Ls, Lepeophtheirus salmonis; Rm, Rhipicephalus microplus; Hc, Haemonchus contortus; Cl, Canis lupus familiaris) show that the M2 to M3 region is highly conserved even between different phyla. Both mutations A301S and T350 M are highlighted in yellow and blue, respectively, in the DmR2-GABA sequence
Fig. 3. Lotilaner is a potent antagonist of the DmS-GABA receptor. a Current traces from a cumulative exposure to increasing dosage of lotilaner obtained for a Xenopus oocyte expressing DmS-GABA. The bars indicate the time period of GABA (10 μM) application. The grey triangle represents the gradual exposure to lotilaner with the respective concentration in μM indicated above. b Averaged inhibition concentration-response curves measured for dieldrin (black circle), fipronil (black triangle, dashed line), lotilaner (black square), and AHC-2229544 (white square) obtained from oocytes expressing DmS-GABA. Individual curves were standardized to the fitted maximal current amplitude and subsequently averaged. Mean ± SEM of experiments carried out with at least four oocytes from two batches each is shown
Fig. 4. Co-application protocol on DmS-GABA. Current traces obtained from X. laevis oocytes expressing DmS-GABA receptors. The left panel shows superimposed traces recorded after the fifth exposure to 1 μM GABA (doted-line trace) and the first and second co-application (black and grey trace, respectively) with 100 nM (a) fipronil, (b) lotilaner, or (c) AHC-2229544 at 1 min interval. The doted-line traces show the fourth and fifths response to 1 μM GABA application. The right panel shows the forth and fifth GABA applications (doted-line) while the black and grey traces represent the first and the second to fifth co-application with 100 nM of (a) fipronil, (b) lotilaner, or (c) AHC-2229544, respectively. The bars indicate the time period of GABA application (interrupted line) or GABA co-applied with the compound (solid line)
Fig. 5. Lotilaner function as a non-competitive antagonist of DmS-GABA receptors. Concentration-response plots for GABA alone (black circle), GABA in the presence of 0.1 μM lotilaner (black triangle), and GABA in the presence of 1 μM lotilaner (black square). EC50 and Rmax values were 9.29 ± 0.81 μM and 97.706 ± 2.45% (n = 5) for GABA, 6.33 ± 0.20 μM and 50.98 ± 0.47% (n = 5) in 0.1 μM lotilaner, and 6.27 ± 1.85 μM and 13.65 ± 1.12% (n = 4) in 1 μM lotilaner. Individual curves were standardized to an initial 100 μM GABA application and subsequently averaged. Mean ± SEM is shown
Fig. 6. Lotilaner antagonism is not affected by mutation causing dieldrin and fipronil resistance. a Current traces obtained from a X. laevis oocyte expressing DmR2-GABA receptors. The interrupted trace shows the fifths response to 1 μM GABA application representing the baseline. The black and grey traces (first and second recording, respectively) have been obtained after oocyte exposure to 1 μM GABA co-applied with 100 nM fipronill. The bars indicate the time period of GABA application (interrupted line) or GABA co-applied with fipronil (solide line). b Averaged inhibition concentration-response curves measured for dieldrin (black circle), fipronil (black triangle, dashed line) and lotilaner (black square), obtained from oocytes expressing DmR2-GABA. Individual curves were standardized to the fitted maximal current amplitude and subsequently averaged. Mean ± SEM of experiments carried out with at least four oocytes from two batches each is shown
Fig. 7. Lotilaner is a potent antagonist of invertebrate GABACl receptors. Averaged inhibition concentration-response curves for lotilaner and fipronil measured on oocytes expressing Ls-GABA1 (white circle and black triangle, dashed line) or Rm-GABA (black square and white diamond, dashed line) as well as on Cl-GABAA α1β2γ2 (white square) for lotilaner. Individual curves were standardized to the fitted maximal current amplitude and subsequently averaged. Mean ± SEM of experiments carried out with at least four oocytes from two batches each is shown
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