Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Simultaneous Measurement and Distribution Analysis of Urinary Nicotine, Cotinine, Trans-3'-Hydroxycotinine, Nornicotine, Anabasine, and Total Nicotine Equivalents in a Large Korean Population.

Measurement of multiple nicotine metabolites and total nicotine equivalents (TNE) might be a more reliable strategy for tobacco exposure verification than measuring single urinary cotinine alone. We simultaneously measured nicotine, cotinine, 3-OH cotinine, nornicotine, and anabasine using 19,874 urine samples collected from the Korean National Health and Nutrition Examination Survey. Of all samples, 18.6% were positive for cotinine, 17.4% for nicotine, 17.3% for nornicotine, 17.6% for 3-OH cotinine, and 13.2% for anabasine. Of the cotinine negative samples, less than 0.3% were positive for all nicotine metabolites, but not for anabasine (5.7%). The agreement of the classification of smoking status by cotinine combined with nicotine metabolites was 0.982-0.994 (Cohen's kappa). TNE3 (the molar sum of urinary nicotine, cotinine, and 3-OH cotinine) was most strongly correlated with cotinine compared to the other nicotine metabolites; however, anabasine was less strongly correlated with other biomarkers. Among anabasine-positive samples, 30% were negative for nicotine or its metabolites, and 25% were undetectable. Our study shows that the single measurement of urinary cotinine is simple and has a comparable classification of smoking status to differentiate between current smokers and non-smokers relative to the measurement of multiple nicotine metabolites. However, measurement of multiple nicotine metabolites and TNE3 could be useful for monitoring exposure to low-level or secondhand smoke exposure and for determining individual differences in nicotine metabolism. Geometric or cultural factors should be considered for the differentiation of tobacco use from patients with nicotine replacement therapy by anabasine.

UPLC-HRESI-MS and GC-MS analysis of the leaves of Nicotiana glauca.

The alkaloid-rich fraction obtained by fractionation of the crude methanolic extract of the leaves of wild tobacco tree Nicotiana glauca Graham (Solanaceae) was analyzed using UPLC-MS and GC-MS. Anabasine, a piperidine alkaloid, was identified as the major constituent with approximately 60 % (m/m) of the alkaloid-rich fraction. In addition to anabasine, six secondary metabolites were identified using high-resolution UPLC-MS. Anabasine was quantified in the leaves to be 1 mg g-1 dry plant material. The GC-MS analysis revealed five compounds with anabasine as the major component, while nicotine was not detected. Moreover, GC-MS was used for the analysis of the volatile oil that was obtained by hydro-distillation from the leaves of N. glauca. The volatile plant oil was found to be rich in oxygenated sesquiterpenes (e.g., ß-bisabolol) and carboxylic acids and esters (e.g., ethyl linoleate and hexadecanoic acid), whereas anabasine was not detected.

The phosphorylated regulator of chemotaxis is crucial throughout biofilm biogenesis in Shewanella oneidensis.

The core of the chemotaxis system of Shewanella oneidensis is made of the CheA3 kinase and the CheY3 regulator. When appropriated, CheA3 phosphorylates CheY3, which, in turn, binds to the rotor of the flagellum to modify the swimming direction. In this study, we showed that phosphorylated CheY3 (CheY3-P) also plays an essential role during biogenesis of the solid-surface-associated biofilm (SSA-biofilm). Indeed, in a ΔcheY3 strain, the formation of this biofilm is abolished. Using the phospho-mimetic CheY3D56E mutant, we showed that CheY-P is required throughout the biogenesis of the biofilm but CheY3 phosphorylation is independent of CheA3 during this process. We have recently found that CheY3 interacts with two diguanylate cyclases (DGCs) and with MxdA, the c-di-GMP effector, probably triggering exopolysaccharide synthesis by the Mxd machinery. Here, we discovered two additional DGCs involved in SSA-biofilm development and showed that one of them interacts with CheY3. We therefore propose that CheY3-P acts together with DGCs to control SSA-biofilm formation. Interestingly, two orthologous CheY regulators complement the biofilm defect of a ΔcheY3 strain, supporting the idea that biofilm formation could involve CheY regulators in other bacteria.

(S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor.

Nematode parasites infect ∼2 billion people world-wide. Infections are treated and prevented by anthelmintic drugs, some of which act on nicotinic acetylcholine receptors (nAChRs). There is an unmet need for novel therapeutic agents because of concerns about the development of resistance. We have selected Asu-ACR-16 from a significant nematode parasite genus, Ascaris suum, as a pharmaceutical target and nicotine as our basic moiety (EC50 6.21 ± 0.56 µM, Imax 82.39 ± 2.52%) to facilitate the development of more effective anthelmintics. We expressed Asu-ACR-16 in Xenopus oocytes and used two-electrode voltage clamp electrophysiology to determine agonist concentration-current-response relationships and determine the potencies (EC50s) of the agonists. Here, we describe the synthesis of a novel agonist, (S)-5-ethynyl-anabasine, and show that it is more potent (EC50 0.14 ± 0.01 µM) than other nicotine alkaloids on Asu-ACR-16. Agonists acting on ACR-16 receptors have the potential to circumvent drug resistance to anthelmintics, like levamisole, that do not act on the ACR-16 receptors.

Simultaneous determination of eight neonicotinoid insecticide residues and two primary metabolites in cucumbers and soil by liquid chromatography-tandem mass spectrometry coupled with QuEChERS.

A new, rapid, sensitive, precise and validated high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of eight neonicotinoid insecticides with their two primary metabolites in cucumbers and soil based on QuEChERS as a pretreatment procedure. In QuEChERS procedure, cucumber samples were extracted with acetonitrile and cleaned using (C18 sorbent material), while soil samples were extracted with a mixture of acetonitrile:dichloromethane (8.3:16.7v:v). The LC-MS/MS conditions were optimized to provide good selectivity and specificity of the developed method where neonicotinoids were separated using gradient elution of water and acetonitrile both containing 0.1% formic acid with Gemini C18 column where the last compound was eluted at 9.5min. Average recoveries of the eight neonicotinoids and their metabolites ranged between 81.6% and 95.7% in fortified cucumber samples with relative standard deviations (RSDs) lower than 13.18% and between 80.3% and 104% in fortified soil samples with relative standard deviations (RSDs) lower than 8.44%. The limits of detection (LODs) and quantification (LOQs) for the ten compounds were in the ranges of (0.08-6.06ng/g) and (0.26-20ng/g), respectively. The method was applied successfully to determine residues and rate of disappearance of the eight neonicotinoids from cucumber and soil and their half-lives where a safety pre-harvest interval of 5days for acetampirid, 12days for imidacloprid, 15days for nitenpyram, 12days for thiamethoxam, 5days for flonicamid, 8days for clothianidin, 2days for Dinotefuran, and 1day for thiacloprid were suggested.

Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

The only African wild tobacco, Nicotiana africana: alkaloid content and the effect of herbivory.

Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory.

Reference interval determination for anabasine: a biomarker of active tobacco use.

Laboratory detection of nicotine exposure is important for establishing eligibility for organ transplant and elective surgery. Nicotine testing is also used to verify compliance with nicotine replacement therapies (NRT), smoking cessation programs and for life insurance purposes. Nicotine metabolites, such as cotinine and trans-3'-hydroxycotinine, are used as biomarkers of nicotine exposure. For some clinical applications, it is important to distinguish between active use of tobacco products versus NRT. Anabasine is a tobacco alkaloid that has been used as a biomarker of active tobacco use. However, the use of anabasine as an insecticide, and its presence in consumables other than nicotine products, suggests that anabasine may not be specific to tobacco use/exposure. Here, we determine the reference interval for anabasine in the urine of nonsmokers and compare it to the range of anabasine concentrations observed in the presence or absence of nicotine metabolites.

A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites.

BACKGROUND: Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies. METHODS: This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min. RESULTS: The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of >0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of <15.0% on average. CONCLUSIONS: The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.

Qualitative profiling and quantification of neonicotinoid metabolites in human urine by liquid chromatography coupled with mass spectrometry.

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.

RNAi-mediated down-regulation of ornithine decarboxylase (ODC) impedes wound-stress stimulation of anabasine synthesis in Nicotiana glauca.

Unlike most Nicotiana species, leaf tissues of the globally significant weed Nicotiana glauca Grah. (Argentinian tree tobacco) contains anabasine as the main component of its alkaloid pool, with concentrations typically increasing several fold in response to wounding of plants. The Δ(1)-piperidinium ring of anabasine is synthesised from cadaverine, via the decarboxylation of lysine, however the identity of the protein catalysing this reaction remains unknown. Recent studies indicate that ornithine decarboxylase (ODC), an enzyme involved in the synthesis of the diamine putrescine, may also possess LDC activity. Previously we found that ODC transcript is markedly up-regulated in leaves of N. glauca in response to wounding. In order to examine the role of ODC in the synthesis of anabasine in N. glauca, transcript levels were constitutively down-regulated in hairy root cultures and transgenic plants via the introduction of a CaMV35S driven ODC-RNAi construct. In addition to the anticipated marked reduction in nicotine concentrations, demonstrating that the ODC-RNAi construct was functioning in vivo, we observed that N. glauca ODC-RNAi hairy root cultures had a significantly diminished capacity to elevate anabasine synthesis in response to treatment with the wound-associated hormone methyl jasmonate, when compared to vector-only controls. We observed also that ODC-RNAi transgenic plants had significantly reduced ability to increase anabasine concentrations following removal of the plant apex. We conclude that ODC does have an important role in enabling N. glauca to elevate levels of anabasine in response to wound-associated stress.

Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): application to the design of related α7 selective ligands.

AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH(2) functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used.

Unique neonicotinoid binding conformations conferring selective receptor interactions.

Neonicotinoid agonists selectively act on the insect nicotinic acetylcholine receptor (nAChR). The molecular basis for this specificity is deciphered by comparisons of two acetylcholine binding proteins (AChBPs) with distinct pharmacological profiles that serve as structural homologues for the nAChR subtypes. Aplysia AChBP has high neonicotinoid sensitivity, whereas Lymnaea AChBP has low neonicotinoid sensitivity, pharmacologies reminiscent of insect and vertebrate nAChR subtypes, respectively. The ligand-receptor interactions for these AChBPs were established by chemical and structural neurobiology approaches. Neonicotinoids and nicotinoids bind in a single conformation with Aplysia AChBP, wherein the electronegative nitro or cyano pharmacophore of the neonicotinoid faces in a reversed orientation relative to the cationic nicotinoid functionality. For Lymnaea AChBP, the neonicotinoids have two binding conformations in this vertebrate receptor model, which are completely inverted relative to each other, whereas nicotinoids are nestled in only one conserved conformation. Therefore, the unique binding conformations of nicotinic agonists determine the selective receptor interactions.

A computational study of the binding of 3-(arylidene) anabaseines to two major brain nicotinic acetylcholine receptors and to the acetylcholine binding protein.

Nicotinic acetylcholine receptors (nAChRs) have become targets for drug development in recent years. 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA), which selectively stimulates the alpha7 nAChR, has been shown to alleviate some cognitive deficits associated with schizophrenia. In this paper we report an analysis of the interactions between 47 arylidene-anabaseines (including 45 benzylidene-anabaseines) and rat brain alpha7 and alpha4beta2 nicotinic acetylcholine receptors, using three different modeling techniques, namely 2D-QSAR, 3D-QSAR and molecular docking to the Aplysia californica acetylcholine binding protein (AChBP), a water soluble, homomeric nAChR surrogate receptor with a known crystal structure. Our investigation indicates the importance of: (1) the nitrogen atom of the tetrahydropyridyl (THP) ring for hydrogen bond formation; (2) pi-pi interactions between the aromatic rings of the ligands and the nAChBP binding site; (3) molecular surface recognition expressed in terms of steric complimentarity. On the basis of the 3D-QSAR results, bulky substituents at positions 2 (and due to the rotational freedom also at position 6) and 4 of the benzylidene moiety, with highly electronegative atoms projecting approximately 3-3.5A away from the benzylidene ring at position 4 seem optimal for enhancing binding affinity to the alpha7 nAChR.

Refined methodology for the determination of neonicotinoid pesticides and their metabolites in honey bees and bee products by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

An analytical method was refined for the extraction and determination of neonicotinoid pesticide residues and their metabolites in honey bees and bee products. Samples were extracted with 2% triethylamine (TEA) in acetonitrile (ACN) followed by salting out, solid phase extraction (SPE) cleanup, and detection using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated in triplicate at three fortification concentrations in each matrix. Good recoveries were observed for most analytes and ranged between 70 and 120% with relative standard deviations between replicates of <20% in most cases. The method limits of detection were 0.2 ng/g for the parent neonicotinoid pesticides and ranged between 0.2 and 15 ng/g for the neonicotinoid metabolites. This refined method provides lower detection limits and improved recovery of neonicotinoids and their metabolites, which will help researchers evaluate subchronic effects of these pesticides, address data gaps related to colony collapse disorder (CCD), and determine the role of pesticides in pollinator decline.

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor.

The pentameric acetylcholine-binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the alpha7 receptor, 3-(2,4-dimethoxybenzylidene)-anabaseine and its 4-hydroxy metabolite, and an indole-containing partial agonist, tropisetron, were solved at 2.7-1.75 A resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist-protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full-length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing alpha7-selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.

Differential regulation of receptor activation and agonist selectivity by highly conserved tryptophans in the nicotinic acetylcholine receptor binding site.

We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (alpha7 Tyr188 or alpha4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the alpha7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in alpha4beta2 and alpha7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human alpha7 and alpha4beta2 and expressed the receptors in Xenopus laevis oocytes. Alpha7 receptors with Trp55 mutated to Gly or Tyr became less responsive to 4OH-GTS-21, whereas mutation of the homologous Trp57 in beta2 to Gly, Tyr, Phe, or Ala resulted in alpha4beta2 receptors that showed increased responses to 4OH-GTS-21. Mutation of alpha7 Trp55 to Val resulted in receptors for which the partial agonist 4OH-GTS-21 became equally efficacious as ACh, whereas alpha4beta2 receptors with the homologous mutation remained nonresponsive to 4OH-GTS-21. In contrast to the striking alterations in agonist activity profiles that were observed with mutations of alpha7 Trp55 and beta2 Trp57, mutations of alpha7 Trp149 or alpha4 Trp154 universally resulted in receptors with reduced function. Our data support the hypothesis that some conserved residues in the nAChR LBD differentially regulate receptor activation by subtype-selective agonists, whereas other equally well conserved residues play fundamental roles in receptor activation by any agonist. Residues like alpha7 Trp149 (alpha4 Trp154) may be considered pillars upon which basic receptor function depends, whereas alpha7 Trp55 (beta2 Trp57) and alpha7 Tyr188 (alpha4 Tyr195) may be fulcra upon which agonists may operate differentially in specific receptor subtypes, consistent with the hypothesis that ACh and 4OH-GTS-21 are able to activate nAChR in distinct ways.

Diverse actions and target-site selectivity of neonicotinoids: structural insights.

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO(2) group of imidacloprid and 2) neonicotinoid-unique stacking and CH-pi bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-alpha nAChR subunits.

Neonicotinoid substituents forming a water bridge at the nicotinic acetylcholine receptor.

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.