Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Divergent Nine-Step Syntheses of Perhydrohistrionicotoxin Analogs and Their Inhibition Activity Toward Chicken α4ß2-Neuronal Nicotinic Acetylcholine Receptors.

Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4ß2-neuronal nAChRs that were more potent than pHTX.

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-ß-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Ligand-gated ion channels as targets of neuroactive insecticides.

The Cys-loop superfamily of ligand-gated ion channels (Cys-loop receptors) is one of the most ubiquitous ion channel families in vertebrates and invertebrates. Despite their ubiquity, they are targeted by several classes of pesticides, including neonicotinoids, phenylpyrazols, and macrolides such as ivermectins. The current commercialized compounds have high target site selectivity, which contributes to the safety of insecticide use. Structural analyses have accelerated progress in this field; notably, the X-ray crystal structures of acetylcholine binding protein and glutamate-gated Cl channels revealed the details of the molecular interactions between insecticides and their targets. Recently, the functional expression of the insect nicotinic acetylcholine receptor (nAChR) has been described, and detailed evaluations using the insect nAChR have emerged. This review discusses the basic concepts and the current insights into the molecular mechanisms of neuroactive insecticides targeting the ligand-gated ion channels, particularly Cys-loop receptors, and presents insights into target-based selectivity, resistance, and future drug design.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dα1/Dß1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dß1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dß1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dß1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dß1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dß1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dß1 orthosteric site over the Dα1-Dα1 orthosteric site.

STAT3 inhibitory activity of naphthoquinones isolated from Tabebuia avellanedae.

The extract of Tabebuia avellanedae has been used as a folk medicine, and the various biological activities of T. avellanedae have been extensively studied. However, few studies have reported which natural products play a role in their biological effects. In this study, we evaluated representative naphthoquinones isolated from T. avellanedae and found that furanonaphthoquinones were the key structures required to exhibit STAT3 phosphorylation inhibitory activities. Our SAR analysis indicated that removal of a hydroxyl group enhanced the STAT3 phosphorylation inhibitory activity. In addition, the combined results of a mobility shift assay, SH2 domain binding assay, and docking simulation by Autodock 4.2.6 suggested that (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (1) could directly bind to the hinge region of STAT3.

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73.

PURPOSE: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity. METHODS: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72. RESULTS: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment. CONCLUSIONS: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.

Availability of NHS-biotin labeling to identify free protein lysine revealed by experiment and MD simulation.

It is known that the crystallizability of protein molecules may be improved by replacing their surface lysine residues with other residue types. Here an experimental method to identify surface lysine residues by NHS-biotin chemical modification combined with MALDI-TOF MS was proposed and was evaluated using PH1033 protein from Pyrococcus horikoshii. Interestingly, the biotinylation experiment with a protein-reagent molar ratio of 1:1 revealed that only seven of twenty-two lysine residues in the protein comprising 144 residues were labeled. To investigate the result, we analyzed structures from a molecular-dynamics simulation mimicking the experiment. A logistic regression analysis revealed that the biotinylation was significantly correlated with four factors relevant to the local environment of lysine residues: the solvent accessibility, the electrostatic energy, the number of hydrogen bonds, and the estimated pKa value. This result is overall in agreement with that from the same analysis on the crystal structure. However, reflecting the flexibility of the protein molecule in solution state, the factors except for the electrostatic energy were highly variable in the MD structures depending upon the protonation state of Tyr87. The present procedure of biotin-labeling can avoid lysine residues with extensive intramolecular interactions that are incompatible with the rational design of protein crystals.

Combined effects of mutations in loop C and the loop D-E-G triangle on neonicotinoid interactions with Drosophila Dα1/chicken ß2 hybrid nAChRs.

Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken ß2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.

Action of six pyrethrins purified from the botanical insecticide pyrethrum on cockroach sodium channels expressed in Xenopus oocytes.

Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds' in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.

Splice Variants of pH-Sensitive Chloride Channel Identify a Key Determinant of Ivermectin Sensitivity in the Larvae of the Silkworm Bombyx mori.

The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl-1 gene from the brain, third thoracic ganglion (T3G), and midgut of larvae of silkworm Bombyx mori It was found that >50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half-maximal activation at a pH of 7.87, and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori γ-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

Comparative ecotoxicity of imidacloprid and dinotefuran to aquatic insects in rice mesocosms.

There are growing concerns about the impacts of neonicotinoid insecticides on ecosystems worldwide, and yet ecotoxicity of many of these chemicals at community or ecosystem levels have not been evaluated under realistic conditions. In this study, effects of two neonicotinoid insecticides, imidacloprid and dinotefuran, on aquatic insect assemblages were evaluated in experimental rice mesocosms. During the 5-month period of the rice-growing season, residual concentrations of imidacloprid were 5-10 times higher than those of dinotefuran in both soil and water. Imidacloprid treatment (10kg/ha) reduced significantly the populations of, Crocothemis servilia mariannae and Lyriothemis pachygastra nymphs, whereas those of Orthetrum albistylum speciosum increased slightly throughout the experimental period. However, Notonecta triguttata, which numbers were high from the start, later declined, indicating possible delayed chronic toxicity, while Guignotus japonicus disappeared. In contrast, dinotefuran (10kg/ha) did not decrease the populations of any species, but rather increased the abundance of some insects, particularly Chironominae spp. larvae and C. servilia mariannae nymphs, with the latter being 1.7x higher than those of controls. This was an indirect effect resulting from increased prey (e.g., chironomid larvae) and lack of competition with other dragonfly species. The susceptibilities of dragonfly nymphs to neonicotinoids, particularly imidacloprid, were consistent with those reported elsewhere. In general, imidacloprid had higher impacts on aquatic insects compared to dinotefuran.

Probing new components (loop G and the α-α interface) of neonicotinoid binding sites on nicotinic acetylcholine receptors.

Neonicotinoid insecticides interact with the orthosteric site on the extracellular ligand binding domain (LBD) of nicotinic acetylcholine receptors (nAChRs), typically activating the cation permeable ion channels. In nAChRs consisting of two α and three non-α subunits, LBDs contain six loops (loops A, B and C on the α subunit and loops D, E and F on the non-α subunit) which make up the orthosteric binding site at the α/non-α subunit interfaces. Recently, an additional site (loop G) on the ß1 strand has been identified. Also, when the α/non-α subunit ratio is 3/2, another binding site is generated at the interface of two adjacent α subunits. Roles for loop G and the α-α interface in the interactions with neonicotinoids are discussed with reference to recent structural and physiological data.

A SNP in the ABCC11 gene is the determinant of human earwax type.

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G --> A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G --> A SNP is the first example of DNA polymorphism determining a visible genetic trait.

Studies on an acetylcholine binding protein identify a basic residue in loop G on the ß1 strand as a new structural determinant of neonicotinoid actions.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the ß1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the α7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.