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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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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)CF 3 moiety is primarily recognized by the β subunit-face in the ligand-binding pocket (interface between α and β 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 β subunit employing a recombinant hybrid nAChR consisting of aphid Mpα2 and rat Rβ2 subunits (with a mutation at T77 on the Rβ2). 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/Mpβ1 interface structural model, thereby underscoring a distinction in its binding mechanism from IMI.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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 IC 50 = 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., (© 2018 Society of Chemical Industry.)

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 17β-estradiol production which can be attributed to an up-regulation of 17β-hydroxysteroid dehydrogenase. Moreover, TCOH exhibited significant antiestrogenic activity with a RIC 20 (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.

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 [ 3 H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [ 3 H]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 α4β2 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.

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 (K ads ) 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 K ads 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 K ads for glucose of at most 0.18 M -1 , which can be compared with K ads for the phenolics investigated here, which fell in the range of 443-42 639 M -1 . The actual value of K ads for glucose may be much closer to zero given the lack of observed glucose uptake with NU-1000 as adsorbent.

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., (Copyright © 2016. Published by Elsevier Inc.)

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 Ca 2+ 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 Q o site of the cytochrome bc 1 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 (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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.

The anticholinesterase (antiChE) organophosphorus (OP) and methylcarbamate (MC) insecticides have been used very effectively as contact and systemic plant protectants for seven decades. About 90 of these compounds are still in use - the largest number for any insecticide chemotype or mode of action. In both insects and mammals, AChE inhibition and acetylcholine accumulation leads to excitation and death. The cholinergic system of insects is located centrally (where it is protected from ionized OPs and MCs) but not at the neuromuscular junction. Structural differences between insect and mammalian AChE are also evident in their genomics, amino acid sequences and active site conformations. Species selectivity is determined in part by inhibitor and target site specificity. Pest population selection with OPs and MCs has resulted in a multitude of modified AChEs of altered inhibitor specificity some conferring insecticide resistance and others enhancing sensitivity. Much of the success of antiChE insecticides results from a suitable balance of bioactivation and detoxification by families of CYP450 oxidases, hydrolases, glutathione S-transferases and others. Known inhibitors for these enzymes block detoxification and enhance potency which is particularly important in resistant strains. The current market for OPs and MCs of 19% of worldwide insecticide sales is only half of that of 10 years ago for several reasons: there have been no major new compounds for 30 years; resistance has eroded their effectiveness; human toxicity problems are still encountered; the patents have expired reducing the incentive to update registration packages; alternative chemotypes or control methods have been developed. Despite this decline, they still play a major role in pest control and the increasing knowledge on their target sites and metabolism may make it possible to redesign the inhibitors for insensitive AChEs and to target new sites in the cholinergic system. The OPs and MCs are down but not out., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells., (Copyright © 2011 Wiley Periodicals, Inc.)

This structure-activity relationship study for neonicotinoids with an N-haloacetylimino pharmacophore identifies several candidate compounds showing outstanding insecticidal potency and consequently leads to establishing their molecular recognition at an insect nicotinic receptor structural model, wherein the neonicotinoid halogen atoms (fluorine, chlorine, bromine, and iodine) variously interact with the receptor loops C-D interfacial niche via H-bonding and/or hydrophobic interactions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In enzymes, the electronic and steric environments of active centres, and therefore their activity in biological processes, are controlled by the surrounding amino acids. In a similar manner, organic ligands have been used for the 'passivation' of metal clusters, that is, inhibition of their aggregation and control of their environment. However, the ability of enzymes to maintain large degrees of accessibility has remained difficult to mimic in synthetic systems in which little room, if any, is typically left to bind to other species. Here, using calix[4]arene macrocycles bearing phosphines as crude mimics of the rigid backbones of proteins, we demonstrate the synthesis of gold clusters and the control of their accessibility through an interplay between the sizes of the calixarene ligands and metal cores. For 0.9-nm cores, 25% of all the gold atoms within the cluster bind to the chemisorption probe 2-naphthalenethiol. This accessibility dramatically decreases with 1.1-nm and 4-nm gold cores.

Bis-imidacloprid (bis-IMI) analogues with suitable alkylene spacers have plant-systemic insecticidal properties. The alkylene-tethered bis-IMI binds in a unique mode to the insect nicotinic acetylcholine receptor (nAChR) wherein the chloropyridine moieties are embraced by two distinct and distant domains. The heptamethylene spacer optimally bridges these two subsites, yet the linker itself binds in a relatively nonspecific manner. This investigation examines the hypothesis that a bis-IMI analogue with a heteroaromatic tether, which undergoes specific interaction(s) with the newly recognized receptor cavity, may enhance the potency relative to those of the alkylene-tethered derivatives. Remarkably, a novel bis-IMI with a furan-2,5-dimethylene fulcrum showed highest receptor potency and insecticidal activity among the analogues with various chemotype spacers. The nAChR structural model, simulating the binding site interactions of the furan-2,5-dimethylene-tethered bis-IMI, reveals that the furan ring is nestled in a hydrophobic pocket, consisting of three aromatic amino acids, and is stabilized via hydrogen bonding.

Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl- and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl- and chlorothiazolyl-neonicotinoids induce SA responses associated with enhanced stress tolerance.

Substituted-phenoxycarbonylimino neonicotinoid ligands with an electron-donating group showed significantly higher affinity to the insect nicotinic receptor relative to that of the analogue with an electron-withdrawing substituent, thereby establishing in silico binding site interaction model featuring that the phenoxy ring of neonicotinoids and the receptor loop D tryptophan indole plane form a face-to-edge aromatic interaction., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The bis-pharmacophore approach applied to neonicotinoid insecticides reveals high binding affinity for heptamethylene bis-N(3),N(3')-imidacloprid fitting a nicotinic acetylcholine receptor model wherein the chloropyridine moieties contact loops E and F and the alkylene linker bridges these two distant domains.

Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tyrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.

Neonicotinoid insecticides are extensively used for crop protection. The chloropyridinyl or chlorothiazolyl nitrogen and tetrahydrofuryl oxygen atoms of neonicotinoids serve as hydrogen acceptors at the target site. This investigation designs and prepares neonicotinoid probes to understand the structure-activity relationships (SARs) at the target site focusing on the water-mediated ligand-protein interactions. 2-Nitroiminoimidazolidine analogues with hydrogen-acceptor N-CH(2)CH(2)CH(2)F and N-CH(2)CH(2)C(O)CH(3) substituents showed higher binding affinities to the Drosophila melanogaster nicotinic receptor than probes with different hydrogen-bonding points in location and capability, suggesting that the appropriately positioned fluorine or carbonyl oxygen plays an important role on hydrogen-bond formation. Their binding site interactions were predicted using a crystal structure of the acetylcholine binding protein. The fluorine or carbonyl oxygen forms a water bridge to Ile-118 (and/or Ile-106) at the binding domain, consistent with that of neonicotinoids with a chloropyridinylmethyl, chlorothiazolylmethyl, or tetrahydrofurylmethyl moiety. Therefore, the present SAR study on binding site interactions helps design potent neonicotinoids with novel substituents.

Molecular interactions of neonicotinoid insecticides with the nicotinic acetylcholine receptor have been mapped by chemical and structural neurobiology approaches, thereby encouraging the biorational design of novel nicotinic ligands. This investigation designs, prepares, and evaluates the target site potency of neonicotinoid analogues with various types of electronegative pharmacophores and subsequently predicts their molecular recognition in the ligand-binding pocket. The N-nitroimino (NNO2) neonicotinoid pharmacophore is systematically replaced by N-nitrosoimino (NNO), N-formylimino [NC(O)H], N-alkyl- and N-arylcarbonylimino [NC(O)R], and N-alkoxy- and N-aryloxycarbonylimino [NC(O)OR] variants. The NNO analogues essentially retain the binding affinity of the NNO2 compounds, while the isosteric NC(O)H congeners have diminished potency. The NC(O)R and NC(O)OR analogues, where R is methyl, trifluoromethyl, phenyl, or pyridin-3-yl, have moderate to high affinities. Orientation of the tip oxygen plays a critical role for binding of the NNO and NC(O)H pharmacophores, and the extended NC(O)R and NC(O)OR moieties are embraced by unique binding domains.

Neonicotinoid agonists with a nitroimino or cyanoimino pharmacophore are the newest of the four most important classes of insecticides. Our studies on the nicotinic receptor structure in the neonicotinoid-bound state revealed a unique niche of about 6 A depth beyond the nitro oxygen or cyano nitrogen tip. The N-substituted imino pharmacophore was therefore extended to fill the gap. Excellent target site selectivity with high insecticidal activity and low toxicity to mammals were achieved rivaling those of the current neonicotinoid insecticides as illustrated here by 3-(6-chloropyridin-3-ylmethyl)-2-trifluoroacetyliminothiazoline and its pyrazinoylimino analogue.

The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.e., Lymnaea stagnalis (Ls) AChBP of low neonicotinoid and high nicotinoid sensitivities and Aplysia californica (Ac) AChBP of high neonicotinoid sensitivity] mimicking vertebrate and insect nAChR subtypes, respectively. The structural basis of subtype selectivity was examined here by photoaffinity labeling. Two azidoneonicotinoid probes in the Ls-AChBP surprisingly modified two distinct and distant subunit interface sites: loop F Y164 of the complementary or (-)-face subunit and loop C Y192 of the principal or (+)-face subunit, whereas three azidonicotinoid probes derivatized only Y192. Both the neonicotinoid and nicotinoid probes labeled Ac-AChBP at only one position at the interface between loop C Y195 and loop E M116. These findings were used to establish structural models of the two AChBP subtypes. In the Ac-AChBP, the neonicotinoids and nicotinoids are nestled in similar bound conformations. Intriguingly, for the Ls-AChBP, the neonicotinoids have two bound conformations that are inverted relative to each other, whereas nicotinoids appear buried in only one conserved conformation as seen for the Ac-AChBP subtype. Accordingly, the subtype selectivity is based on two disparate bound conformations of nicotinic agonists, thereby establishing an atypical concept for neonicotinoid versus nicotinoid selectivity between insect and vertebrate nAChRs.

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.

Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid and thiacloprid, are nonprotonated and selective for insects and their nicotinic receptors, whereas nicotinoids such as nicotine and epibatidine are cationic and selective for mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), as a structural surrogate for the extracellular ligand-binding domain of the nicotinic receptor, is similarly sensitive to neonicotinoids and nicotinoids. It therefore seemed possible that the proposed very different interactions of the neonicotinoids and nicotinoids might be examined with a single AChBP by using optimized azidochloropyridinyl photoaffinity probes. Two azidoneonicotinoids with a nitro or cyano group were compared with the corresponding desnitro or descyano azidonicotinoids. The four photoactivated nitrene probes modified AChBP with up to one agonist for each subunit based on analysis of the intact derivatized protein. Identical modification sites were observed by collision-induced dissociation analysis for the neonicotinoids and nicotinoids with similar labeling frequency of Tyr-195 of loop C and Met-116 of loop E at the subunit interface. The nitro- or cyano-substituted guanidine/amidine planes of the neonicotinoids provide a unique electronic conjugation system to interact with loop C Tyr-188. The neonicotinoid nitro oxygen and cyano nitrogen contact loop C Cys-190/Ser-189, whereas the cationic head of the corresponding nicotinoids is inverted for hydrogen-bonding and cation-pi contact with Trp-147 and Tyr-93. These structural models based on AChBP directly map the elusive neonicotinoid binding site and further describe the molecular determinants of agonists on nicotinic receptors.

The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.

Serine hydrolase KIAA1363 is highly expressed in invasive cancer cells and is the major protein in mouse brain diethylphosphorylated by and hydrolyzing low levels of chlorpyrifos oxon (CPO) (the activated metabolite of a major insecticide). It is also the primary CPO-hydrolyzing enzyme in spinal cord, kidney, heart, lung, testis, and muscle but not liver, a pattern of tissue expression confirmed by fluorophosphonate-rhodamine labeling. KIAA1363 gene deletion using homologous recombination reduces CPO binding, hydrolysis, and metabolism 3-29-fold on incubation with brain membranes and homogenates determined with 1 nM [(3)H-ethyl]CPO and the inhibitory potency for residual CPO with butyrylcholinesterase as a biomarker. Studies with knockout mice further show that KIAA1363 partially protects brain AChE and monoacylglycerol lipase from CPO-induced in vivo inhibition. Surprisingly, mouse brain KIAA1363 and AChE are similar in in vitro sensitivity to seven methyl, ethyl, and propyl but not higher alkyl OP insecticides and analogues, prompting structural comparisons of the active sites of KIAA1363 and AChE relative to OP potency and selectivity. Homology modeling based largely on the Archaeoglobus fulgidus esterase crystal structure indicates that KIAA1363 has a catalytic triad of S191, D348, and H378, a GDSAG motif, and an oxyanion hole of H113, G114, G115, and G116. Excellent selectivity for KIAA1363 is achieved on OP structure optimization with long alkyl chain substituents suggesting that KIAA1363 has larger acyl and leaving group pockets than those of AChE. KIAA1363 reactivates faster than AChE presumably due to differences in the uncoupling of the catalytic triad His upon phosphorylation. The structural modeling of KIAA1363 helps us understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.

Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

6'-Methylpyrido[3,4-b]norhomotropane [synthesis as the racemate reported here] is more potent at the alpha4beta2 nicotinic receptor than any previous bridged nicotinoid. The two nitrogens and 6'-methyl substituent are superimposable on the two nitrogens and 6-chloro substituent of epibatidine, with the best fit on comparing the chair conformer of the (1R)-pyridonorhomotropane with natural (1R)-epibatidine. In this pharmacophore model, the 6'-methyl substituent may be equivalent to the acetyl methyl of acetylcholine.

Fipronil [5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulfinylpyrazole] is one of the most important insecticides. Structure-activity studies described here reveal that fipronil retains its very high binding potency at the human beta3 and house fly gamma-aminobutyric acid (GABA) receptors and toxicity to house flies on replacing the pyrazole trifluoromethylsulfinyl moiety with tert-butyl or isopropyl and the phenyl trifluoromethyl substituent with ethynyl, trifluoromethoxy, bromo or chloro. Among the compounds studied, those with other alkyl groups at the 4-position of the pyrazole, as well as phenyl substitution without one or both of the 2,6-dichloro groups, are less effective. 5-Amino-4-tert-butyl-3-cyano-1-(2,6-dichloro-4-ethynylphenyl)pyrazole is highly effective and almost isosteric with 4-tert-butyl-3-cyano-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2]octane (the most potent 4-alkyl-1-phenyltrioxabicyclooctane) as a noncompetitive GABA antagonist and insecticide. These findings are interpreted as three binding subsites in the GABA receptor: a hydrophobic site undergoing steric interaction with the tert-butyl or equivalent group; a hydrogen bonding site to pyrazole N-2; a pi bonding site to the face of the phenyl moiety; with supplemental enhancement by the 3-cyano and 4-ethynyl substituents.

Simple alcohols and ethers have been studied with the MM4 force field. The structures of 13 molecules have been well fit using the MM4 force field. Moments of inertia have been fit with rms percentage errors as indicated: 18 moments for ethers, 0.28%; 21 moments for alcohols, 0.22%. Rotational barriers and conformational equilibria have also been examined, and the experimental and ab initio results are reproduced substantially better with MM4 than they were with MM3. Much of the improvement comes from the use of additional interaction terms in the force constant matrix, of which the torsion-bend and torsion-torsion are particularly important. Induced dipoles are included in the calculation, and dipole moments are reasonably well fit. It has been possible for the first time to fit conformational energetic data for both open chain and cyclic alcohols (e.g., propanol and cyclohexanol) with the same parameter set. For vibrational spectra, over a total of 82 frequencies, the rms error is 27 cm(-1), as opposed to 38 cm(-1) with MM3. Both the alpha and beta bond shortening resulting from the presence of the electronegative oxygen atom in the molecule are well reproduced. The electronegativity of the oxygen is sufficient that one must also include not only the alpha and beta electronegativity effects on bond lengths, but also on angle distortions, if structures are to be well reproduced. The heats of formation of 32 alcohols and ethers were fit overall to within experimental error (weighted standard deviation error 0.26 kcal/mol)., (Copyright 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1447-1472, 2003)

The anomeric effect has been studied for a variety of compounds using the MM4 force field, and also using MP2/6-311++G(2d,2p) ab initio calculations and experimental data for reference purposes. Geometries and energies, including conformational, rotational barriers, and heats of formation were examined. Overall, the agreement of MM4 with the experimental and ab initio data is good, and significantly better than the agreement obtained with the MM3 force field. The anomeric effect is represented in MM4 by various explicit terms in the force constant matrix. The bond length changes are accounted for with torsion-stretch elements. The angle changes are accounted for with torsion-bend elements. The energies are taken into account with a number of torsional terms in the usual way. A torsion-torsion interaction is also of some importance. With all of these elements included in the calculation, the MM4 results now appear to be adequately accurate. The heats of formation were examined for a total of 12 anomeric compounds, and the experimental values were fit by MM4 with an RMS error of 0.42 kcal/mol., (Copyright 2003 Wiley Periodicals, Inc. J Comput Chem 24: 1473-1489, 2003)

Cation-pi interaction, a prominent feature in agonist recognition by neurotransmitter-gated ion channels, does not apply to the anomalous action of neonicotinoids at the insect nicotinic acetylcholine receptor (nAChR). Insect-selective neonicotinoids have an electronegative pharmacophore (tip) in place of the ammonium or iminium cation of the vertebrate-selective nicotinoids, suggesting topological divergence of the agonist-binding sites in insect and vertebrate nAChRs. This study defines the molecular and electronic basis for the potent and selective interaction of the neonicotinoid electronegative pharmacophore with a unique subsite of the Drosophila but not of the vertebrate alpha4beta2 nAChR. Target site potency and selectivity are retained when the usual neonicotinoid N-nitroimine (=NNO(2)) electronegative tip is replaced with N-nitrosoimine (=NNO) or N-(trifluoroacetyl)imine (=NCOCF(3)) in combination with an imidazolidine, imidazoline, thiazolidine, or thiazoline heterocycle. X-ray crystallography establishes coplanarity between the heterocyclic and imine planes, including the electronegative substituent in the trans configuration. The functional tip is the coplanar oxygen atom of the N-nitrosoimine or the equivalent oxygen of the N-nitroimine. Quantum mechanics in the gas and aqueous phases fully support the conserved coplanarity and projection of the strongly electronegative tip. Further, a bicyclic analogue with a nitro tip in the cis configuration but retaining coplanarity has a high potency, whereas the N-trifluoromethanesulfonylimine (=NSO(2)CF(3)) moiety lacking coplanarity confers very low activity. The coplanar system between the electronegative tip and guanidine-amidine moiety extends the conjugation and facilitates negative charge (delta(-)) flow toward the tip, thereby enhancing interaction with the proposed cationic subsite such as lysine or arginine in the Drosophila nAChR.

Objective: To determine whether older people who volunteered for a health intervention study used the health care system differently from a randomly selected group of eligible non-volunteers., Methods: Three hundred sixty-three volunteers and 92 non-volunteers were compared; both groups were members of a large health maintenance organization (HMO)., Results: Volunteers were more educated and had been members of the HMO longer. Volunteers used the health care system significantly more at all time periods, but their cost per contact was significantly lower than that for non-volunteers. Non-volunteers were more likely to have a comorbid condition and had fewer arthritis-related health care contacts., Conclusions: There will always be problems in generalizing results of studies with volunteers, but volunteers are, nevertheless, the most appropriate controls if the experimental participants are also volunteers. Designs should ideally include both volunteer and non-volunteer controls.