Cartereau A et al. (2024), Pharmacology and molecular modeling studies of ...

XB-ART-61023

Toxicol Appl Pharmacol 2024 Oct 10;492:117123. doi: 10.1016/j.taap.2024.117123.

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Pharmacology and molecular modeling studies of sulfoxaflor, flupyradifurone and neonicotinoids on the human neuronal α7 nicotinic acetylcholine receptor.

Cartereau A , Bouchouireb Z , Kaaki S , Héricourt F , Taillebois E , Le Questel JY , Thany SH .

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We conducted electrophysiological and molecular docking studies using a heterologous expression system (Xenopus oocytes) to compare the effects of four neonicotinoids (acetamiprid, imidacloprid, clothianidin and thiamethoxam), one sulfoximine, (sulfoxaflor), and one butenolide (flupyradifurone), on human α7 neuronal nicotinic acetylcholine receptors (nAChRs). All neonicotinoids (except thiamethoxam), as well as the recently introduced nAChR competitive modulators, flupyradifurone and sulfoxaflor, appear to be weaker agonists than acetylcholine. Two mutations in loop C (E211N and E211P) and one mutation in loop D (Q79K), known to be involved in the binding properties of neonicotinoids were introduced to the α7 wild type. Interestingly, the acetylcholine and nicotine-evoked activation was not modified in human α7 mutated receptors, but the net charge was enhanced for clothianidin and imidacloprid, respectively. Flupyradifurone responses strongly increased under the Q79K mutation. The molecular docking investigations demonstrated that the orientations and interactions of the ligands considered were in accordance with those observed experimentally. Specifically, the charged fragments of acetylcholine and nicotine, used as reference ligands, and their neonicotinoid homologs were found to be surrounded by aromatic residues, with key interactions with Trp171 and Y210. Furthermore, the molecular docking investigations predicted the water-mediated interaction between the carbonyl oxygen of acetylcholine and the Nsp2 nitrogen of the pyridine ring for nicotine (as well as for the majority of the corresponding neonicotinoid fragments) and main chain NH of L141. The docking scores, extending over a significant range of 6 kcal/mol, showed that most neonicotinoids were poorly stabilized in the α7 nAChR compared to acetylcholine, except sulfoxaflor.

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Species referenced: Xenopus laevis
Genes referenced: ace
GO keywords: acetylcholine receptor activity

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	Graphical abstract
	Fig. 1. Amino acid sequences of the α7WT and α7 mutated receptors and chemical structure of all tested compounds. (a) Protein alignments of the N-terminal domain from α7WT and mutants of nAChRs. The mutations are indicated in red. Mutation Q79K is in loop D and the mutations E211P and E211N are located in loop C. Loops involved in ligand binding are highlighted with blue boxes. Sites of cysteine residues involved in the cys-loop are marked with asterisks and the vicinal residues characteristic of the α-type subunit are boxed. (b) Chemical structures of acetylcholine, nicotine and all tested insecticides: clothianidin, acetamiprid, imidacloprid, thiamethoxam, sulfoxaflor and flupyradifurone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. Agonist actions of acetylcholine, nicotine, neonicotinoid insecticides and their derivatives on human α7WT receptors. (a) Concentration response curves of acetylcholine (ACh), nicotine (Nic), neonicotinoids: acetamiprid (ACE), clothianidin (CLT), imidacloprid (IMI), thiamethoxam (TMX), and their derivatives: sulfoxaflor (SFX) and flupyradifurone (FLU), obtained on α7WT nAChRs expressed in X. laevis oocytes. Each point plotted represents the mean ± S.E.M of four to fifteen experiments (n = 3–7 batches). For each compound, the net charge was normalized to net charge obtained after application of ACh 60 μM and data were fitted to the Hill equation. (b) Representative raw data traces for each compound used at 100 μM. (c) Histograms representing the normalized net charge induced by 12 s application of 100 μM of each compound. Each bar indicates the mean ± S.E.M of eleven to thirteen experiments (n = 5 batches). Mann-Whitney test was used to statistically compare each normalized net charge to ACh 100 μM. Significant differences from ACh are indicated with asterisks (**** indicates p < 0.0001).
	Fig. 3. Agonist actions of acetylcholine, nicotine, neonicotinoid insecticides and their derivatives on human α7E211N receptors. (a) Representative raw data traces for each compound used at 100 μM. (b) Histograms representing the normalized net charge induced by 12 s application of 100 μM of each compound. Each bar indicates the mean ± S.E.M of eleven to thirteen experiments (n = 6 batches). Mann-Whitney test was used to statistically compare each normalized net charge to ACh 100 μM. Significant differences from ACh are indicated with asterisks (* indicates p < 0.05, indicates p < 0.01, * indicates p < 0.001, **** indicates p < 0.0001).
	Fig. 4. Agonist actions of acetylcholine, nicotine, neonicotinoids insecticides and their derivatives on human α7E211P receptors. (a) Representative raw data traces for each compound used at 100 μM. (b) Histograms representing the normalized net charge induced by 12 s application of 100 μM of each compound. Each bar indicates the mean ± S.E.M of eleven to thirteen experiments (n = 5 batches). Mann-Whitney test was used to statistically compare each normalized net charge to ACh 100 μM. Significant differences from ACh are indicated with asterisks (* indicates p < 0.05, indicates p < 0.01, * indicates p < 0.001, **** indicates p < 0.0001).
	Fig. 5. Agonist actions of acetylcholine, nicotine, neonicotinoid insecticides and their derivatives on human α7Q79K receptors. (a) Representative raw data traces for each compound used at 100 μM. (b) Histograms representing the normalized net charge induced by 12 s application of 100 μM of each compound. Each bar indicates the mean ± S.E.M of eleven to thirteen experiments (n = 6 batches). Mann-Whitney test was used to statistically compare each normalized net charge to ACh 100 μM. Significant differences from ACh are indicated with asterisks (* indicates p < 0.05, indicates p < 0.01, * indicates p < 0.001, **** indicates p < 0.0001).
	Fig. 6. Docking predicted binding modes of ACh (a), Nic (b), the nitro‑neonicotinoids IMI (c) CLT (d), and TMX (e), and one cyano‑neonicotinoid ACE (f), with wild type human α7 nAChR. The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks and the various kinds of interactions are indicated with dashed lines: hydrogen-bonds in blue, π stacking in green, π cation in orange and water bridges in purple. The main α7 nAChR residues are identified with their one letter code and numbering. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Fig. 7. Docking predicted binding modes of the two diastereoisomers of SFX taken into account in this work (RS (a) SS (b) and docking predicted binding mode of FLU (c), with wild type human α7 nAChR. The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligand is shown in balls and sticks and the various kinds of interactions are indicated with dashed lines: hydrogen-bonds in blue, π stacking in green, π cation in orange and water bridges in purple. The main α7 nAChR residues are identified with their one letter code and numbering. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
	Figure S1 Sequence alignment showing loops D and C of vertebrate and insect neuronal nicotinic acetylcholine receptor a7 subunits.
	Figure S2 Co-application of acetylcholine with flupyradifurone. Averaged raw data traces for 60 µM acetylcholine (ACh) and 1 µM flupyradifurone (FLU), on a7WT nAChRs (a) and �7E211N (b), �7E211P (c), and �7Q79K (d) receptors. Each bar indicates when the compound is added (12 s). Mann-Whitney test was used to statistically compare normalized net charge from coapplication of ACh 60 µM + FLU 1 µM at ACh 60 µM. Each experiment represents 3 different batches. Significant differences are indicated with asterisks (indicates p < 0.05, *indicates p < 0.01).
	Figure S3 Co-application of acetylcholine with sulfoxaflor. Averaged raw data traces for 60 µM acetylcholine (ACh) and 1 µM sulfoxaflor (SFX), on a7WT nAChRs (a) and �7E211N (b), �7E211P (c), and �7Q79K (d) receptors. Each bar indicates when the compound is added (12 s). Mann-Whitney test was used to statistically compare normalized net charge from coapplication of ACh 60 µM + SFX 1 µM at ACh 60 µM. Each experiment represents 3-4 different batches. ns : no significant differences (p > 0.05).
	Figure S4 Co-application of acetylcholine with both sulfoxaflor and flupyradifurone. Averaged raw data traces for 60 µM acetylcholine (ACh) and 10 µM sulfoxaflor (SFX) + flupyradifurone 10 µM (FLU), on a7WT nAChRs (a), and a7E211N (b), a7E211P (c) and a7Q79K (d) mutations. The n values range between 3 and 4 oocytes. Mann-Whitney was used to statistically compare normalized net charge from co-application of ACh 60 µM +FLU 1 µM at ACh 60 µM. Each experiment represents 3-4 different batches. ns : no significant differences (p > 0.05).
	Figure S5: Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the E211N mutant of human a7 nAChR (the carbon atoms of the N211 residue are colored in pink). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.
	Figure S5: Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the E211N mutant of human a7 nAChR (the carbon atoms of the N211 residue are colored in pink). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.
	Figure S6. Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the E211P mutant of human a7 nAChR (the carbon atoms of the P211 residue are colored in pink). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.
	Figure S6. Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the E211P mutant of human a7 nAChR (the carbon atoms of the P211 residue are colored in pink). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.
	Figure S7. Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the Q79K mutant of human a7 nAChR (the carbon atoms of the K79 residue are colored in pink)). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.
	Figure S7. Docking predicted binding modes of ACh a) NIC b) IMI c) CLO d) TMX e) ACE (f) SFX (RS diastereoisomer) g) SFX (SS diastereoisomer) h) and FLU i) with the Q79K mutant of human a7 nAChR (the carbon atoms of the K79 residue are colored in pink)). The main and complementary components of the binding site are colored in blue and yellow, respectively. The ligands are shown in balls and sticks, and the various kinds of interactions are indicated in dashed lines : hydrogen-bonds in blue, p stacking in green, p cation in orange and water bridges in purple. The main a7 nAChR residues are identified with their one letter code and numbering.