Navigating the complexities of docking tools with nicotinic receptors and acetylcholine binding proteins in the realm of neonicotinoids.

Molecular docking, pivotal in predicting small-molecule ligand binding modes, struggles with accurately identifying binding conformations and affinities. This is particularly true for neonicotinoids, insecticides whose impacts on ecosystems require precise molecular interaction modeling. This study scrutinizes the effectiveness of prominent docking software (Ledock, ADFR, Autodock Vina, CDOCKER) in simulating interactions of environmental chemicals, especially neonicotinoid-like molecules with nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding proteins (AChBPs). We aimed to assess the accuracy and reliability of these tools in reproducing crystallographic data, focusing on semi-flexible and flexible docking approaches. Our analysis identified Ledock as the most accurate in semi-flexible docking, while Autodock Vina with Vinardo scoring function proved most reliable. However, no software consistently excelled in both accuracy and reliability. Additionally, our evaluation revealed that none of the tools could establish a clear correlation between docking scores and experimental dissociation constants (Kd) for neonicotinoid-like compounds. In contrast, a strong correlation was found with drug-like compounds, bringing to light a bias in considered software towards pharmaceuticals, thus limiting their applicability to environmental chemicals. The comparison between semi-flexible and flexible docking revealed that the increased computational complexity of the latter did not result in enhanced accuracy. In fact, the higher computational cost of flexible docking with its lack of enhanced predictive accuracy, rendered this approach useless for this class of compounds. Conclusively, our findings emphasize the need for continued development of docking methodologies, particularly for environmental chemicals. This study not only illuminates current software capabilities but also underscores the urgency for advancements in computational molecular docking as it is a relevant tool to environmental sciences.

Corrigendum: Cloning and expression of cockroach α7 nicotinic acetylcholine receptor subunit.

[This corrects the article DOI: 10.3389/fphys.2020.00418.].

The calcium-calmodulin-dependent protein kinase kinase inhibitor, STO-609, inhibits nicotine-induced currents and intracellular calcium increase in insect neurosecretory cells.

Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates and are considered to be major targets of neonicotinoid insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, in order to explore the intracellular mechanisms leading to the regulation of insect neuronal nAChRs in more detail. Using whole-cell patch-clamp and fura-2AM calcium imaging techniques, we found that a novel CaMKK/AMPK pathway could be involved in the intracellular regulation of DUM neuron nAChRs. The CaMKK selective inhibitor, STO, reduced nicotinic current amplitudes, and strongly when co-applied with α-Bgt. Interestingly, intracellular application of the AMPK activator, A-76, prevented the reduction in nicotine-induced currents observed in the presence of the AMPK inhibitor, dorsomorphin. STO prevented the increase in intracellular calcium induced by nicotine, which was not dependent on α-Bgt. Currents induced by 1 mM LMA, a selective activator of nAChR2, were reduced under bath application of STO, and mecamylamine, which blocked nAChR2 subtype, inhibited the increase in intracellular calcium induced by LMA. These findings provide insight into potential complex mechanisms linked to the modulation of the DUM neuron nAChRs and CaMKK pathway.

Effect of Acetamiprid, a Neonicotinoid Insecticide, on Locomotor Activity of the American Cockroach.

Toxicological studies have shown that the American cockroach Periplaneta americana (Linnaeus) is a classical model for studying the mode of action of commonly used insecticides. In a previous study, we demonstrated that thiamethoxam and clothianidin decreased locomotor activity in an open-field-like apparatus. Here, we tested the effect of the neonicotinoid acetamiprid when applied orally, topically, or injected into the haemolymph. We found that acetamiprid was also able to impair locomotor activity in the open-field-like apparatus. When treated with acetamiprid, a strong alteration in locomotor activity was observed 1 h, 24 h, and 48 h after haemolymph and topical applications. Oral application induced an impairment of locomotor activity at 24 h and 48 h. A comparison of the present data with our previously published results showed that neonicotinoids were more active when injected into the haemolymph compared to oral and topical applications. These findings increased our understanding of the effect of neonicotinoid insecticides on insect locomotor activity, and demonstrated that the cyano-substituted neonicotinoid, acetamiprid, was able to alter cockroach locomotor activity.

Development of CHARMM compatible force field parameters and molecular dynamics simulations for the pesticide flupyradifurone.

Flupyradifurone (FLU) is a novel butenolide insecticide with partial agonist activity for insect nicotinic acetylcholine receptors. Its safety for non-target organisms has been questioned in the literature, despite initial claims of its harmlessness. Detailed understanding of its toxicity and related molecular mechanisms remain under discussion. Thus, in this work, an optimized set of CHARMM compatible parameters for FLU is presented. CHARMM General Force Field program was used as a starting point while the non-bonded and bonded parameters were adjusted and optimized to reproduce MP2/6-31G(d) accuracy level results. For the validity assessment of these parameters, infrared spectrum, water-octanol partition coefficient, and normal modes were computed and compared to experimental values found in the literature. Several MD simulations of FLU in water and FLU in complex with an acetylcholine-binding protein were performed to estimate the ability of the optimized parameters to correctly describe its torsional space and reproduce observed crystallographic trends respectively.

Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Molecular Mechanism of Action of Neonicotinoid Insecticides.

Since neonicotinoid insecticides were first introduced several years ago, most of them have been banned by the European Union due to their potentially adverse effects on humans and useful insects [...].

Identification of sulfonamide compounds active on the insect nervous system: Molecular modeling, synthesis and biological evaluation.

Insect nicotinic acetylcholine receptors (nAChRs) are a recognized target for insecticide design. In this work, we have identified, from a structure-based approach using molecular modeling tools, ligands with potential selective activity for pests versus pollinators. A high-throughput virtual screening with the Openeye software was performed using a library from the ZINC database, thiacloprid being used as the target structure. The top sixteen molecules were then docked in α6 cockroach and honeybee homomeric nAChRs to check from a theoretical point of view relevant descriptors in favor of pest selectivity. Among the selected molecules, one original sulfonamide compound has afterward been synthesized, together with various analogs. Two compounds of this family have been shown to behave as activators of the cockroach cholinergic synaptic transmission.

Effect of the combination of DEET and flupyradifurone on the tick Ixodes ricinus: Repellency bioassay and pharmacological characterization using microtransplantation of synganglion membranes.

Ticks are vectors of many human and animal pathogens, and represent a major threat to public health. In recent years, an increase in tick-borne diseases has been observed, and new strategies are therefore needed in order to control tick numbers and reduce human tick bites. In the present study, we adapted the previous tick repellency bioassay based on the exploration behavior of the tick, using the ToxTrac software and video-tracking, to compare the repellent effect of two compounds on the tick Ixodes ricinus: N,N-diethyl-methyl-m-toluamide (DEET), and butenolide, flupyradifurone (FLU). We found that when applied alone, 10% DEET or FLU have no/or low repellency effect. But, the combination of both 10% DEET and FLU demonstrated a significant repellency effect against I. ricinus, similar to the repellency of 20% DEET. Using membrane microtransplantation, we evaluated the effect of DEET and FLU on native acetylcholine receptors expressed on the tick synganglion. We found that DEET has no effect on acetylcholine-evoked currents, but significantly reduced nicotine-induced current amplitudes. FLU induced an ionic current but was not able to reduce acetylcholine or nicotine evoked currents. The combination of both DEET and FLU strongly reduced nicotine-evoked currents. Finally, we demonstrated that our recording device for repellency, as well as the use of membrane microtransplantation, could be used as methods to study the mode of action of active compounds on ticks.

Impairments in learning and memory performances associated with nicotinic receptor expression in the honeybee Apis mellifera after exposure to a sublethal dose of sulfoxaflor.

Sulfoxaflor is a new insecticide which acts on the nicotinic acetylcholine receptor (nAChRs) in a similar way to neonicotinoids. However, sufloxaflor (SFX) is thought to act in a different manner and is thus proposed as an alternative in crop protection. The goal of this study is to evaluate the toxicity of SFX and its sublethal effect on the honeybee Apis mellifera after acute exposure. In toxicological assay studies, the LD50 value and sublethal dose (corresponding to the NOEL: no observed effect level) were 96 and 15 ng/bee, respectively. Using the proboscis extension response paradigm, we found that an SFX dose of 15 ng/bee significantly impairs learning and memory retrieval when applied 12 h before conditioning or 24 h after olfactory conditioning. SFX had no effect on honeybee olfactory performance when exposure happened after the conditioning. Relative quantitative PCR experiments performed on the six nicotinic acetylcholine receptor subunits demonstrated that they are differently expressed in the honeybee brain after SFX exposure, whether before or after conditioning. We found that intoxicated bees with learning defects showed a strong expression of the Amelß1 subunit. They displayed overexpression of Amelα9 and Amelß2, and down-regulation of Amelα1, Amelα3 and Amelα7 subunits. These results demonstrated for the first time that a sublethal dose of SFX could affect honeybee learning and memory performance and modulate the expression of specific nAChR subunits in the brain.

The use of insecticide mixtures containing neonicotinoids as a strategy to limit insect pests: Efficiency and mode of action.

Synthetic insecticides continue to be the main strategy for managing insect pests, which are a major concern for both crop protection and public health. As nicotinic acetylcholine receptors play a central role in insect neurotransmission, they are the molecular target of neurotoxic insecticides such as neonicotinoids. These insecticides are used worldwide and have shown high efficiency in culture protection. However, the emergence of insect resistance mechanisms, and negative side-effects on non-target species have highlighted the need for a new control strategy. In this context, the use of insecticide mixtures with synergistic effects have been used in order to decrease the insecticide dose, and thus delay the selection of resistance-strains, and limit their negative impact. In this review, we summarize the available data concerning the mode of action of neonicotinoid mixtures, as well as their toxicity to various insect pests and non-target species. We found that insecticide mixtures containing neonicotinoids may be an effective strategy for limiting insect pests, and in particular resistant strains, although they could also negatively impact non-target species such as pollinating insects.

Calmidazolium induces a decrease in nicotine-induced currents and intracellular calcium levels after pulse application of nicotine onto insect neurosecretory cells.

Dorsal unpaired median (DUM) neurons, are a class of insect neurosecretory cells, which are involved in the control of several functions, such as excretion and reproduction, or the release of neurohormones. Previous studies demonstrated that they express different nicotinic acetylcholine receptor subtypes, in particular α-bungarotoxin-insensitive receptors, with nAChR1 and nAChR2 subtypes. Here, we demonstrated that pulse application of 1 mM nicotine (300 ms pulse duration) induced inward currents which were reduced under bath application of 15 µM calmidazolium, a calmodulin inhibitor. Bath application of 0.5 µM α-bungarotoxin had no effect on calmidazolium action, suggesting that it could have an indirect effect through α-bungarotoxin-insensitive receptors. Indeed, nicotine-evoked currents were reduced by 10 µM d-tubocurarine, and completely blocked by 5 µM mecamylamine, which affected nAChR1 and nAChR2 subtypes, respectively. Our results demonstrated that nAChR2 subtypes are involved in the indirect effect of calmidazolium. Moreover, we found that this calmidazolium effect was associated to a strong reduction in intracellular calcium levels after pulse application of 1 mM nicotine. Thus, compared to previous studies on mammalian cells, calmidazolium did not cause an increase in intracellular calcium levels in DUM neurons, suggesting that different calcium mechanisms are involved in the calmidazolium effect.

Mode of Action of Neonicotinoid Insecticides Imidacloprid and Thiacloprid to the Cockroach Pameα7 Nicotinic Acetylcholine Receptor.

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation.

Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 µM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes.

Synergic effect of a quinuclidine benzamide complexed with borane, the LMA10233, in combination with seven pesticides.

Some quinuclidine benzamide compounds have been found to modulate nicotinic acetylcholine receptors in both mammals and insects. In particular, the quaternarization of 3-amino quinuclidine benzamide derivatives with dichloromethane gave charged N-chloromethylated quinuclidine compounds, disclosing an antagonist profile on homomeric α7 nAChRs. Here, we synthesized and studied the toxicological effect of LMA10233, a quinuclidine-borane complex analogue, the LMA10233, on the pea aphid Acyrthosiphon pisum and found that LMA10233 only exhibit proper toxicity on A. pisum larvae when applied in concentrations of over 10 µg/ml. We assessed the ability of LMA10233 to enhance the toxicity of different insecticides. When a sublethal concentration of LMA10233 was combined with the LC10 of each compound, we found a strong increase in toxicity at 24 h and 48 h of exposure for clothianidin, fipronil and chlorpyrifos, and only at 24 h for imidacloprid, acetamiprid and deltamethrin. However, when the pesticide was used at the LC50, only acetamiprid showed a synergistic effect with LMA10233. When the concentration of LMA10233 was decreased, we found that up to 80-90% of mortality was obtained due to the synergism between acetamiprid and LMA10233. No similar effect was observed with other insecticides. We conclude that such quinuclidine-borane complex compounds could increase the toxic effect of insecticides at low concentrations.

An Overview on the Effect of Neonicotinoid Insecticides on Mammalian Cholinergic Functions through the Activation of Neuronal Nicotinic Acetylcholine Receptors.

Neonicotinoid insecticides are used worldwide and have been demonstrated as toxic to beneficial insects such as honeybees. Their effectiveness is predominantly attributed to their high affinity for insect neuronal nicotinic acetylcholine receptors (nAChRs). Mammalian neuronal nAChRs are of major importance because cholinergic synaptic transmission plays a key role in rapid neurotransmission, learning and memory processes, and neurodegenerative diseases. Because of the low agonist effects of neonicotinoid insecticides on mammalian neuronal nAChRs, it has been suggested that they are relatively safe for mammals, including humans. However, several lines of evidence have demonstrated that neonicotinoid insecticides can modulate cholinergic functions through neuronal nAChRs. Major studies on the influence of neonicotinoid insecticides on cholinergic functions have been conducted using nicotine low-affinity homomeric α7 and high-affinity heteromeric α4ß2 receptors, as they are the most abundant in the nervous system. It has been found that the neonicotinoids thiamethoxam and clothianidin can activate the release of dopamine in rat striatum. In some contexts, such as neurodegenerative diseases, they can disturb the neuronal distribution or induce oxidative stress, leading to neurotoxicity. This review highlights recent studies on the mode of action of neonicotinoid insecticides on mammalian neuronal nAChRs and cholinergic functions.

Cloning and Expression of Cockroach α7 Nicotinic Acetylcholine Receptor Subunit.

Understanding insect nicotinic acetylcholine receptor (nAChR) subtypes is of major interest because they are the main target of several insecticides. In this study, we have cloned a cockroach Pameα7 subunit that encodes a 518 amino acid protein with futures typical of nAChR subunit, and sequence homology to α7 subunit. Pameα7 is differently expressed in the cockroach nervous system, in particular in the antennal lobes, optical lobes and the mushroom bodies where specific expression was found in the non-compact Kenyon cells. In addition, we found that cockroach Pameα7 subunits expressed in Xenopus laevis oocytes can assemble to form homomeric receptors. Electrophysiological recordings using the two-electrode voltage clamp method demonstrated that nicotine induced an I max current of -92 ± 27 nA at 1 mM. Despite that currents are low with the endogenous ligand, ACh, this study provides information on the first expression of cockroach α7 homomeric receptor.

Effects of the DAG analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents through α-bungarotoxin-insensitive nicotinic acetylcholine receptors expressed on cockroach neurosecretory cells.

We previously demonstrated that the cockroach α-bungarotoxin-sensitive nicotinic acetylcholine receptors, nAChR1 and nAChR2 subtypes, are differently sensitive to intracellular calcium pathways. Here, using whole cell patch-clamp recordings, we studied the effects of the diacylglycerol (DAG) analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents under an α-bungarotoxin treatment. Our results demonstrated that DiC8 reduced nicotine and clothianidin evoked currents. 10 µM DiC8 suppressed the increase in nicotine-induced currents which was brought about by application of 5 mM caffeine or 9 mM Ca2+, whereas DiC8 did not affect the decrease in nicotine-induced currents induced by BAPTA. Similarly, bath application of caffeine or 9 mM Ca2+ did not change the clothianidin effects, and the amplitude of clothianidin-induced currents was not affected. However, co-application of both 10 µM DiC8 with 9 mM Ca2+, caffeine or BAPTA reduced clothianidin current amplitudes. We conclude that nicotine and clothianidin differently modulate nAChR1 and nAChR2 subtypes under DiC8 treatment, and that nicotine activates nAChR1, whereas clothianidin activates both nAChR1 and nAChR2 subtypes.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid.

Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 µM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 µM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 µM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 µM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 µM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.