Prediction of the Interactions of a Large Number of Per- and Poly-Fluoroalkyl Substances with Ten Nuclear Receptors.

Per- and poly-fluoroalkyl substances (PFASs) are persistent, toxic chemicals that pose significant hazards to human health and the environment. Screening large numbers of chemicals for their ability to act as endocrine disruptors by modulating the activity of nuclear receptors (NRs) is challenging because of the time and cost of in vitro and in vivo experiments. For this reason, we need computational approaches to screen these chemicals and quickly prioritize them for further testing. Here, we utilized molecular modeling and machine-learning predictions to identify potential interactions between 4545 PFASs with ten different NRs. The results show that some PFASs can bind strongly to several receptors. Further, PFASs that bind to different receptors can have very different structures spread throughout the chemical space. Biological validation of these in silico findings should be a high priority.

The vitamin D receptor as a potential target for the toxic effects of per- and polyfluoroalkyl substances (PFASs): An in-silico study.

Due to their persistence and toxicity, perfluoroalkyl and polyfluoroalkyl substances (PFASs) constitute significant hazards to human health and the environment. Their effects include immune suppression, altered hormone levels, and osteoporosis. Recently, the most studied PFAS, perfluorooctanoic acid (PFOA), was shown to competitively binding to the Vitamin D receptor (VDR). VDR plays a crucial role in regulating genes involved in maintaining immune, endocrine, and calcium homeostasis, suggesting it may be a target for at least some of the health effects of PFAS. Hence, this study examined the potential binding of 5206 PFASs to VDR using molecular docking, molecular dynamics, and free energy binding calculations. We identified 14 PFAS that are predicted to interact strongly with VDR, similar to the natural ligands. We further investigated the interactions of VDR with 256 PFASs of established commercial importance. Eighty-three (32%) of these 256 commercially important PFAS were predicted to be stronger binders to VDR than PFOA. At least 16 PFASs of regulatory importance, because they have been identified in water supplies and human blood samples, were also more potent binders to VDR than PFOA. Further, PFASs are usually found together in contaminated drinking water and human blood samples, which raises the concern that multiple PFASs may act together as a mixture on VDR function, potentially producing harmful effects on the immune, endocrine, and bone homeostasis.

Predicting the binding of small molecules to nuclear receptors using machine learning.

Nuclear receptors (NRs) are important biological targets of endocrine-disrupting chemicals (EDCs). Identifying chemicals that can act as EDCs and modulate the function of NRs is difficult because of the time and cost of in vitro and in vivo screening to determine the potential hazards of the 100 000s of chemicals that humans are exposed to. Hence, there is a need for computational approaches to prioritize chemicals for biological testing. Machine learning (ML) techniques are alternative methods that can quickly screen millions of chemicals and identify those that may be an EDC. Computational models of chemical binding to multiple NRs have begun to emerge. Recently, a Nuclear Receptor Activity (NuRA) dataset, describing experimentally derived small-molecule activity against various NRs has been created. We have used the NuRA dataset to develop an ensemble of ML-based models to predict the agonism, antagonism, binding and effector binding of small molecules to nine different human NRs. We defined the applicability domain of the ML models as a measure of Tanimoto similarity to the molecules in the training set, which enhanced the performance of the developed classifiers. We further developed a user-friendly web server named 'NR-ToxPred' to predict the binding of chemicals to the nine NRs using the best-performing models for each receptor. This web server is freely accessible at http://nr-toxpred.cchem.berkeley.edu. Users can upload individual chemicals using Simplified Molecular-Input Line-Entry System, CAS numbers or sketch the molecule in the provided space to predict the compound's activity against the different NRs and predict the binding mode for each.

In Vitro characterization of the endocrine disrupting effects of per- and poly-fluoroalkyl substances (PFASs) on the human androgen receptor.

Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated. In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)- 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)- 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system.

Deciphering the Flupyrimin Binding Surface on the Insect Nicotinic Acetylcholine Receptor.

A novel insecticide flupyrimin (FLP) with a trifluoroacetyl pharmacophore acts as an antagonist at the insect nicotinic acetylcholine receptor (nAChR). This investigation examines a hypothesis that the FLP C(O)CF3 moiety is primarily recognized by the ß subunit-face in the ligand-binding pocket (interface between α and ß subunits) of the insect nAChR. Accordingly, we evaluate the atomic interaction between a fluorine atom of FLP and the partnering amino acid side chain on the ß subunit employing a recombinant hybrid nAChR consisting of aphid Mpα2 and rat Rß2 subunits (with a mutation at T77 on the Rß2). The H-donating T77R, T77K, T77N, or T77Q nAChR enhances the FLP binding potency relative to that of the wild-type receptor, whereas the affinity of neonicotinoid imidaclprid (IMI) with a nitroguanidine pharmacophore remains unchanged. These results facilitate the establishment of the unique FLP molecular recognition at the Mpα2/Mpß1 interface structural model, thereby underscoring a distinction in its binding mechanism from IMI.

Structure-based virtual screening of perfluoroalkyl and polyfluoroalkyl substances (PFASs) as endocrine disruptors of androgen receptor activity using molecular docking and machine learning.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) pose a substantial threat as endocrine disruptors, and thus early identification of those that may interact with steroid hormone receptors, such as the androgen receptor (AR), is critical. In this study we screened 5,206 PFASs from the CompTox database against the different binding sites on the AR using both molecular docking and machine learning techniques. We developed support vector machine models trained on Tox21 data to classify the active and inactive PFASs for AR using different chemical fingerprints as features. The maximum accuracy was 95.01% and Matthew's correlation coefficient (MCC) was 0.76 respectively, based on MACCS fingerprints (MACCSFP). The combination of docking-based screening and machine learning models identified 29 PFASs that have strong potential for activity against the AR and should be considered priority chemicals for biological toxicity testing.

Structure-based discovery of the endocrine disrupting effects of hydraulic fracturing chemicals as novel androgen receptor antagonists.

Hydraulic fracturing (HF) technology is increasingly utilized for oil and gas extraction operations. The widespread use of HF has led to concerns of negative impacts on both the environment and human health. Indeed, the potential endocrine disrupting impacts of HF chemicals is one such knowledge gap. Herein, we used structure-based molecular docking to assess the binding affinities of 60 HF chemicals to the human androgen receptor (AR). Five HF chemicals had relatively high predicted AR binding affinity, suggesting the potential for endocrine disruption. We next assessed androgenic and antiandrogenic activities of these chemicals in vitro. Of the five candidate AR ligands, only Genapol®X-100 significantly modified AR transactivation. To better understand the structural effect of Genapol®X-100 on the potency of AR inhibition, we compared the antiandrogenic activity of Genapol®X-100 with that of its structurally similar chemical, Genapol®X-080. Interestingly, both Genapol®X-100 and Genapol®X-080 elicited an antagonistic effect at AR with 20% relative inhibitory concentrations of 0.43 and 0.89 µM, respectively. Furthermore, we investigated the mechanism of AR inhibition of these two chemicals in vitro, and found that both Genapol®X-100 and Genapol®X-080 inhibited AR through a noncompetitive mechanism. The effect of these two chemicals on the expression of AR responsive genes, e.g. PSA, KLK2, and AR, was also investigated. Genapol®X-100 and Genapol®X-080 altered the expression of these genes. Our findings heighten awareness of endocrine disruption by HF chemicals and provide evidence that noncompetitive antiandrogenic Genapol®X-100 could cause adverse endocrine health effects.

Structural Dynamics of Agonist and Antagonist Binding to the Androgen Receptor.

Androgen receptor (AR) is a steroid hormone nuclear receptor which upon binding its endogenous androgenic ligands (agonists), testosterone and dihydrotestosterone (DHT), alters gene transcription, producing a diverse range of biological effects. Antiandrogens, such as the pharmaceuticals bicalutamide and hydroxyflutamide, act as agonists in the absence of androgens and as antagonists in their presence or in high concentration. The atomic level mechanism of action by agonists and antagonists of AR is less well characterized. Therefore, in this study, multiple 1 µs molecular dynamics (MD), docking simulations, and perturbation-response analyses were performed to more fully explore the nature of interaction between agonist or antagonist and AR and the conformational changes induced in the AR upon interaction with different ligands. We characterized the mechanism of the ligand entry/exit and found that helix-12 and nearby structural motifs respond dynamically in that process. Modeling showed that the agonist and antagonist/agonist form a hydrogen bond with Thr877/Asn705 and that this interaction is absent for antagonists. Agonist binding to AR increases the mobility of residues at allosteric sites and coactivator binding sites, while antagonist binding decreases mobility at these important sites. A new site was also identified as a potential surface for allosteric binding. These results shed light on the effect of agonists and antagonists on the structure and dynamics of AR.

Fiprole insecticide resistance of Laodelphax striatellus: electrophysiological and molecular docking characterization of A2'N RDL GABA receptors.

BACKGROUND: Phenylpyrazole (fiprole) insecticides, including ethiprole, fipronil and flufiprole with excellent activity on rice planthoppers, are very important in Asia but resistance has developed after decades of use. The molecular mechanism of fipronil- but not ethiprole-resistance has been previously studied in rice planthoppers. In our laboratory, a small brown planthopper Laodelphax striatellus strain with ethiprole-resistance was cultured and the molecular mechanisms of ethiprole resistance and of cross-resistance among fiprole insecticides were investigated. RESULTS: Ethiprole-resistant L. striatellus has >5000-fold resistance compared to the susceptible strain, and exhibits around 200-fold cross-resistance with fipronil and flufiprole. RDL genes were isolated from susceptible and ethiprole-resistant L. striatellus and expressed in Xenopus oocytes. Electrophysiological studies showed fiprole insecticides inhibited γ-aminobutyric acid (GABA)-induced current with IC50 = 0.1-1.4 µM to LsRDL-S homomers. In LsRDL-R with A2'N mutation, only 1-13% inhibition was observed on treatment with 10 µM ethiprole, fipronil or flufiprole. Homology models indicate A2'N mutation allows crosslinking hydrogen bonding between Asn sidechains at the 2' position around the channel pore, blocking insecticides from interacting near this position. In contrast, insecticides showed favorable binding near A2' in wild-type L. striatellus. CONCLUSION: Cross-resistance is increasing for fiprole insecticides in L. striatellus and management strategies are necessary to minimize resistance. © 2018 Society of Chemical Industry.

Assessment of the Endocrine-Disrupting Effects of Trichloroethylene and Its Metabolites Using in Vitro and in Silico Approaches.

Trichloroethylene (TCE) is a ubiquitous environmental contaminant, which may have effects on both ecosystem and human health. TCE has been reported to cause several toxic effects, but little effort has been made to assess the ecological risks of TCE or its major metabolites: trichloroethanol (TCOH), trichloroacetic acid, and oxalic acid (OA). In this study, the endocrine-disrupting potential of TCE and its metabolites were investigated using in vitro and in silico approaches. We examined alterations in the steroidogenesis pathway using the NCI-H295R cell line and utilized receptor-mediated luciferase reporter cell lines to identify effects on estrogen and androgen receptors. Molecular docking was also used to explore chemical interactions with these receptors. All test chemicals except OA significantly increased 17ß-estradiol production which can be attributed to an up-regulation of 17ß-hydroxysteroid dehydrogenase. Moreover, TCOH exhibited significant antiestrogenic activity with a RIC20 (20% relative inhibitory concentration) of 3.7 × 10-7 M. Molecular docking simulation supported this finding with lower docking scores for TCOH, indicating that hydrogen bonds may stabilize the interaction between TCOH and the estrogen receptor binding pocket. These findings suggest that TCE contamination poses an endocrine-disrupting threat, which has implications for both ecological and human health.

Flupyrimin: A Novel Insecticide Acting at the Nicotinic Acetylcholine Receptors.

A novel chemotype insecticide flupyrimin (FLP) [N-[(E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [3H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [3H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4ß2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.

Insights into Supramolecular Sites Responsible for Complete Separation of Biomass-Derived Phenolics and Glucose in Metal-Organic Framework NU-1000.

The molecular origins of adsorption of lignin-derived phenolics to metal-organic framework NU-1000 are investigated from aqueous solution as well as in competitive mode with glucose present in the same aqueous mixture. A comparison of adsorption equilibrium constants (Kads) for phenolics functionalized with either carboxylic acid or aldehyde substituents demonstrated only a slight increase (less than a factor of 6) for the former according to both experiments and calculations. This small difference in Kads between aldehyde and carboxylic-acid substituted adsorbates is consistent with the pyrene unit of NU-1000 as the adsorption site, rather than the zirconia nodes, while at saturation coverage, the adsorption capacity suggests multiple guests per pyrene. Experimental standard free energies of adsorption directly correlated with the molecular size and electronic structure calculations confirmed this direct relationship, with the pyrene units as adsorption site. The underlying origins of this relationship are grounded in noncovalent π-π interactions as being responsible for adsorption, the same interactions present in the condensed phase of the phenolics, which to a large extent govern their heat of vaporization. Thus, NU-1000 acts as a preformed aromatic cavity for driving aromatic guest adsorption from aqueous solution and does so specifically without causing detectable glucose adsorption from aqueous solution, thereby achieving complete glucose-phenolics separations. The reusability of NU-1000 during an adsorption/desorption cycle was good, even with some of the phenolic compounds with greatest affinity not easiliy removed with water and ethanol washes at room temperature. A competitive adsorption experiment gave an upper bound for Kads for glucose of at most 0.18 M-1, which can be compared with Kads for the phenolics investigated here, which fell in the range of 443-42 639 M-1. The actual value of Kads for glucose may be much closer to zero given the lack of observed glucose uptake with NU-1000 as adsorbent.

Indole-3-carbinol (I3C) analogues are potent small molecule inhibitors of NEDD4-1 ubiquitin ligase activity that disrupt proliferation of human melanoma cells.

The HECT domain-containing E3 ubiquitin ligase NEDD4-1 (Neural precursor cell Expressed Developmentally Down regulated gene 4-1) is frequently overexpressed in human cancers and displays oncogenic-like properties through the ubiquitin-dependent regulation of multiple protein substrates. However, little is known about small molecule enzymatic inhibitors of HECT domain-containing ubiquitin ligases. We now demonstrate that indole-3-carbinol (I3C), a natural anti-cancer phytochemical derived from cruciferous vegetables such as cabbage and broccoli, represents a new chemical scaffold of small molecule enzymatic inhibitors of NEDD4-1. Using in vitro ubiquitination assays, I3C, its stable synthetic derivative 1-benzyl-I3C and five novel synthetic analogues were shown to directly inhibit NEDD4-1 ubiquitination activity. Compared to I3C, which has an IC50 of 284µM, 1-benzyl-I3C was a significantly more potent NEDD4-1 enzymatic inhibitor with an IC50 of 12.3µM. Compounds 2242 and 2243, the two indolecarbinol analogues with added methyl groups that results in a more nucleophilic benzene ring π system, further enhanced potency with IC50s of 2.71µM and 7.59µM, respectively. Protein thermal shift assays that assess small ligand binding, in combination with in silico binding simulations with the crystallographic structure of NEDD4-1, showed that each of the indolecarbinol compounds bind to the purified catalytic HECT domain of NEDD4-1. The indolecarbinol compounds inhibited human melanoma cell proliferation in a manner that generally correlated with their effectiveness as NEDD4-1 enzymatic inhibitors. Taken together, we propose that I3C analogues represent a novel set of anti-cancer compounds for treatment of human melanomas and other cancers that express indolecarbinol-sensitive target enzymes.

Pesticide Chemical Research in Toxicology: Lessons from Nature.

Pesticide researchers are students of nature, and each new compound and mechanism turns a page in the ever-expanding encyclopedia of life. Pesticides are both probes to learn about life processes and tools for pest management to facilitate food production and enhance health. In contrast to some household and industrial chemicals, pesticides are assumed to be hazardous to health and the environment until proven otherwise. About a thousand current pesticides working by more than 100 different mechanisms have helped understand many processes and coupled events. Pesticide chemical research is a major source of toxicology information on new natural products, novel targets or modes of action, resistance mechanisms, xenobiotic metabolism, selective toxicity, safety evaluations, and recommendations for safe and effective pest management. Target binding site models help define the effect of substituent changes and predict modifications for enhanced potency and safety and circumvention of resistance. The contribution of pesticide chemical research in toxicology is illustrated here with two each of the newer or most important insecticides, herbicides, and fungicides. The insecticides are imidacloprid and chlorantraniliprole acting on the nicotinic acetylcholine receptor and the ryanodine receptor Ca2+ channel, respectively. The herbicides are glyphosate that inhibits aromatic amino acid biosynthesis and mesotrione that prevents plastoquinone and carotenoid formation. The fungicides are azoxystrobin inhibiting the Qo site of the cytochrome bc1 complex and prothioconazole inhibiting the 14α-demethylase in ergosterol biosynthesis. The two target sites involved for each type of pesticide account for 27-40% of worldwide sales for all insecticides, herbicides, and fungicides. In each case, selection for resistance involving a single amino acid change in the binding site or detoxifying enzyme circumvents the pesticide chemists's structure optimization and guarantees survival of the pest and a continuing job for the design chemist. These lessons from nature are a continuing part of pest management and maintaining human and environmental health.

Circulating tumour DNA profiling reveals heterogeneity of EGFR inhibitor resistance mechanisms in lung cancer patients.

Circulating tumour DNA (ctDNA) analysis facilitates studies of tumour heterogeneity. Here we employ CAPP-Seq ctDNA analysis to study resistance mechanisms in 43 non-small cell lung cancer (NSCLC) patients treated with the third-generation epidermal growth factor receptor (EGFR) inhibitor rociletinib. We observe multiple resistance mechanisms in 46% of patients after treatment with first-line inhibitors, indicating frequent intra-patient heterogeneity. Rociletinib resistance recurrently involves MET, EGFR, PIK3CA, ERRB2, KRAS and RB1. We describe a novel EGFR L798I mutation and find that EGFR C797S, which arises in ∼33% of patients after osimertinib treatment, occurs in <3% after rociletinib. Increased MET copy number is the most frequent rociletinib resistance mechanism in this cohort and patients with multiple pre-existing mechanisms (T790M and MET) experience inferior responses. Similarly, rociletinib-resistant xenografts develop MET amplification that can be overcome with the MET inhibitor crizotinib. These results underscore the importance of tumour heterogeneity in NSCLC and the utility of ctDNA-based resistance mechanism assessment.

Novel GABA receptor pesticide targets.

The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use of safe and effective pest control agents.

Selective molecular recognition by nanoscale environments in a supported iridium cluster catalyst.

The active sites of enzymes are contained within nanoscale environments that exhibit exquisite levels of specificity to particular molecules. The development of such nanoscale environments on synthetic surfaces, which would be capable of discriminating between molecules that would nominally bind in a similar way to the surface, could be of use in nanosensing, selective catalysis and gas separation. However, mimicking such subtle behaviour, even crudely, with a synthetic system remains a significant challenge. Here, we show that the reactive sites on the surface of a tetrairidium cluster can be controlled by using three calixarene-phosphine ligands to create a selective nanoscale environment at the metal surface. Each ligand is 1.4 nm in length and envelopes the cluster core in a manner that discriminates between the reactivities of the basal-plane and apical iridium atoms. CO ligands are initially present on the clusters and can be selectively removed from the basal-plane sites by thermal dissociation and from the apical sites by reactive decarbonylation with the bulky reactant trimethylamine-N-oxide. Both steps lead to the creation of metal sites that can bind CO molecules, but only the reactive decarbonylation step creates vacancies that are also able to bond to ethylene, and catalyse its hydrogenation.

Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist.

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [(3)H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [(3)H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site.

Neuroactive insecticides: targets, selectivity, resistance, and secondary effects.

Neuroactive insecticides are the principal means of protecting crops, people, livestock, and pets from pest insect attack and disease transmission. Currently, the four major nerve targets are acetylcholinesterase for organophosphates and methylcarbamates, the nicotinic acetylcholine receptor for neonicotinoids, the γ-aminobutyric acid receptor/chloride channel for polychlorocyclohexanes and fiproles, and the voltage-gated sodium channel for pyrethroids and dichlorodiphenyltrichloroethane. Species selectivity and acquired resistance are attributable in part to structural differences in binding subsites, receptor subunit interfaces, or transmembrane regions. Additional targets are sites in the sodium channel (indoxacarb and metaflumizone), the glutamate-gated chloride channel (avermectins), the octopamine receptor (amitraz metabolite), and the calcium-activated calcium channel (diamides). Secondary toxic effects in mammals from off-target serine hydrolase inhibition include organophosphate-induced delayed neuropathy and disruption of the cannabinoid system. Possible associations between pesticides and Parkinson's and Alzheimer's diseases are proposed but not established based on epidemiological observations and mechanistic considerations.