Search

Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B 1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode ( Haemonchus contortus ) and silkworm ( Bombyx mori ) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B 1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls.  [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) receptors are important targets of parasiticides/insecticides. Several 4-substituted analogs of the partial GABA A receptor agonist 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) were synthesized and examined for their antagonism of insect GABA receptors expressed in Drosophila S2 cells or Xenopus oocytes. Thio-4-PIOL showed weak antagonism of three insect GABA receptors. The antagonistic activity of Thio-4-PIOL was enhanced by introducing bicyclic aromatic substituents into the 4-position of the isothiazole ring. The 2-naphthyl and the 3-biphenylyl analogs displayed antagonist potencies with half maximal inhibitory concentrations in the low micromolar range. The 2-naphthyl analog induced a parallel rightward shift of the GABA concentration–response curve, suggesting competitive antagonism by these analogs. Both compounds exhibited weak insecticidal activities against houseflies. Thus, the orthosteric site of insect GABA receptors might be a potential target site of insecticides. [ABSTRACT FROM AUTHOR]

Highlights: [•] GluCl/GABACl mRNAs and proteins are predominantly expressed in the adult head. [•] GluCl immunostaining was found in the optic lobe, leg motor neuron, and femur. [•] The tissue distribution of GluCls differs from that of GABACls. [•] GluCls and GABACls may have distinct physiological roles. [ABSTRACT FROM AUTHOR]

Abstract: Invertebrate γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors. [Copyright &y& Elsevier]

Abstract: A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl)butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100μM in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100μM, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10μM in these receptors, although the inhibition at 100μM was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500μM using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site. [Copyright &y& Elsevier]

Abstract: Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [3H]MLM A4 were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P316S mutant channels were 37- and 100-fold less sensitive to MLM A4 when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the β10 strand, the M2–M3 linker, and the M3 region influence MLM A4 binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A4. [Copyright &y& Elsevier]

Octopamine (OA) is a biogenic amine that controls a variety of important physiological processes and behaviors of invertebrates. To identify the amino acid residues interacting with (R)-OA in a β-adrenergic-like OA receptor from the silkworm Bombyx mori (BmOAR2), the wild-type receptor and seven mutant receptors with an amino acid substitution at a potential orthosteric site were expressed in HEK-293 cells and examined for their ability to elevate intracellular cAMP levels ([cAMP]i) in response to (R)-OA. The S206A mutant receptor retained the ability to increase [cAMP]i after (R)-OA treatment. In contrast, the other six mutant receptors (D115A, S202A, Y300F, Y300N, Y300L, and Y300A) lacked the ability to elevate [cAMP]i. These results indicate that Asp115, Ser202, and Tyr300 participate in (R)-OA binding and the activation of BmOAR2. Homology modeling studies suggest that Ser202 and Tyr300 interact with the phenolic OH group of (R)-OA, whereas Asp115 interacts with the β-OH group and the NH2 group of (R)-OA. [ABSTRACT FROM AUTHOR]

Abstract: A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [3H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca2+ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological properties. [Copyright &y& Elsevier]

Abstract: Tyramine (TA) is a biogenic amine in invertebrates. cDNA encoding the TA receptor (TAR) BmTAR2 was cloned from the nerve tissue of the silkworm Bombyx mori. The receptor''s functional and pharmacological properties were examined in BmTAR2-transfected HEK-293 cells. In [3H]TA binding assays, BmTAR2 showed considerably higher affinity for TA than for other biogenic amines, with an IC50 value of 57.5 nM. Moreover, TA induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in cells, with an EC50 value of 11.6 nM, whereas octopamine and dopamine increased [Ca2+]i only at concentrations above 100 μM. A few antagonists were found to inhibit the TA-induced increases in [Ca2+]i; the rank order of potency was yohimbine > chlorpromazine > mianserin. TA showed no effect on intracellular cAMP concentration. The data indicate that BmTAR2 belongs to the second class of TARs, which are selectively coupled to intracellular Ca2+ mobilization. RT-PCR analysis revealed that BmTAR2 was expressed predominantly in the nervous tissue of B. mori larvae, suggesting that TA has neurotransmitter and neuromodulatory roles that are mediated by BmTAR2. [Copyright &y& Elsevier]

Abstract: Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues. We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10−7 M with EC50 of 3.30 nM, while BmDopR2 required 10−6 M DA for activation. In BmDopR1-expressing cells, DA at 10−6–10−4 M induced 30–50% lower cAMP production than 10−7 M DA. BmDopR2-expressing cells showed a standard sigmoidal dose–response, with maximum cAMP levels attained with 10−5–10−4 M DA and EC50 of 1.30 μM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics. [Copyright &y& Elsevier]

Abstract: The γ-aminobutyric acid (GABA) receptor bears important sites of action for insecticides. Alantrypinone is an insecticidal alkaloid that acts as a selective antagonist for housefly (vs rat) GABA receptors, and is considered to be a lead compound for the development of a safer insecticide. In an attempt to obtain compounds with greater activity, a series of racemic alantrypinone derivatives were systematically synthesized using hetero Diels–Alder reactions, and a total of 34 compounds were examined for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate, a high-affinity non-competitive antagonist, to housefly-head membranes. The assay results showed that (1) there is no significant difference between the potencies of natural (+)-alantrypinone and its synthetic racemate; (2) the amide NHs at the 2- and 18-positions are important for high activity; (3) there is a considerable drop in potency for compounds without an aromatic ring at the 16-position; and (4) a large substituent at the 3-position is detrimental to high activity. [Copyright &y& Elsevier]

Abstract: A number of widely diverse compounds that show inhibitory activities at the picrotoxinin binding sites in housefly and rat GABA receptors were investigated by using the distance comparison technique (DISCOtech) and comparative molecular field analysis (CoMFA) methods to explore the pharmacophore models and the three-dimensional quantitative structure–activity relationships (3D-QSAR) of the compounds. These compounds consist of three diverse types of noncompetitive GABA receptor antagonists, i.e., trioxabicyclooctanes and their derivatives, picrodendrins and related terpenoids, and fipronil and its analogs. For investigation of the structural requirements for inhibitory activity at the picrotoxinin binding site of GABA receptor, DISCOtech pharmacophore models containing one center of hydrophobic ring and two hydrogen bond acceptor atoms for both housefly-head and rat-brain GABA receptors were constructed, respectively. In particular, the interacting areas in housefly receptors appear to be wider than that in rat receptors, the differences between rat and housefly receptor models implicate the selectivity of noncompetitive GABA receptor antagonists. In addition, corresponding CoMFA models with good statistical indices (r2 >0.9 and q 2 >0.5) were also obtained. These models can be used as guidance for the development of new compounds with high activities and selectivities. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to identify noncompetitive γ-aminobutyric acid receptor (GABAR) antagonists that are effective in nematodes, as well as to examine the hypothesis that the noncompetitive antagonism of the GABAR underlies the nematocidal activity of quassinoids against free-living nematodes of the Diplogastridae family. First, 14 known GABAR antagonists were screened for the effectiveness of their nematocidal activity in Diplogastridae. As a result, 3-isopropyl-1-phospha-2,6,7-trioxabicyclo[2.2.2]octane 1-sulfides (3-isopropyl-BPTs) were found to have high nematocidal activities, and 4-cyclohexyl-3-isopropyl-BPT (23) and 3-isopropyl-4-(2-propenyl)-BPT (27) were the two most potent analogues; these compounds are equipotent to samaderine B and more potent than the anthelmintic abamectin. 23-resistant nematodes, selected by challenge with 23, showed cross-resistance to samaderine B. 23 (10μM) reduced [3H]23 binding to nematode membranes by 30.4%. Samaderine B (10μM) resulted in a similar level of the inhibition, but had neither additive nor synergistic effects on the 23 inhibition of [3H]23 binding. These findings suggest that samaderine B shares a common binding site with the GABAR antagonist 23 in Diplogastridae. The results of comparative molecular field analysis, a method of three-dimensional quantitative structure–activity relationship analysis, supported this conclusion. [Copyright &y& Elsevier]

Thirteen seco-prezizaane terpenoids isolated from star anise species (Illcium floridanum, Illcium parviflorum, and Illcium verum) were investigated for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, to housefly-head and rat-brain membranes. Veranisatin A was found to be the most potent inhibitor in both membranes, with an IC50fly of 78.5 nM and an IC50rat of 271 nM, followed by anisatin (IC50fly=123 nM; IC50rat=282 nM). Six of the other 11 tested compounds were effective only in housefly-head membranes. Pseudoanisatin proved to display a high (>26-fold) selectivity for housefly versus rat GABA receptors (IC50fly=376 nM; IC50rat >10,000 nM). Although pseudoanisatin does not structurally resemble EBOB, Scatchard plots indicated that the two compounds bind to the same site in housefly receptors. Anisatin and pseudoanisatin exhibited moderate insecticidal activity against German cockroaches. Comparative molecular field analysis (CoMFA), a method of three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis, demonstrated that seco-prezizaane terpenoids can bind to the same site as do picrotoxane terpenoids such as picrotoxinin and picrodendrins, and the CoMFA maps allowed us to identify the parts of the molecules essential to high activity in housefly GABA receptors. [Copyright &y& Elsevier]

γ-Aminobutyric acid (GABA) receptors (GABARs) are ligand-gated Cl− channels, which cause an influx of Cl− that inhibits excitation in postsynaptic cells upon activation. GABARs are important targets for drugs and pest control chemicals. We previously reported that the isoxazoline ectoparasiticide fluralaner inhibits GABA-induced currents in housefly (Musca domestica) GABARs by binding to the putative binding site in the transmembrane subunit interface. In the present study, we investigated whether fluralaner inhibits the GABA response in the GABAR activated state, the resting state, or both, using two-electrode voltage clamp electrophysiology protocols. We found that inhibition progresses over time to steady-state levels by repeated short applications of GABA during fluralaner perfusion. The GABA response was not impaired by fluralaner treatment in the GABAR resting state. However, once inhibited, the GABA response was not restored by repeated applications of GABA. These findings suggest that fluralaner might reach the binding site of the activated conformation of GABARs in a stepwise fashion and tightly bind to it. [Display omitted] • Fluralaner, an isoxazoline ectoparasiticide, potently inhibits GABARs. • Inhibition by fluralaner progresses over time after every GABAR activation. • Fluralaner does not inhibit the GABA response in the GABAR resting state. • Fluralaner hardly dissociates from the binding site upon GABAR activation. [ABSTRACT FROM AUTHOR]

(R)-Octopamine (OA), a major invertebrate biogenic amine, plays an important role in a wide variety of physiological processes as a neurohormone, neuromodulator, and neurotransmitter in insects. OA receptors (OARs) are class A G protein-coupled receptors that specifically bind OA to activate downstream signaling cascades by coupling to G proteins and presumably other regulatory proteins. These receptors are broadly classified as α- and β-adrenergic-like OARs (α- and β-ALOARs). OARs are considered important targets of insecticides and acaricides. In the present study, we examined the actions of an array of 13 heterocyclic OAR agonists with the moieties that correspond to the phenyl group and the basic nitrogen atom of OA on α- and β-ALOARs from the silkworm (Bombyx mori) and the signaling pathways activated through these actions. The results indicated that these compounds display structure-dependent receptor subtype selectivity and G protein subtype preference, underscoring the need to determine which subtype and signaling pathway mediates toxicologically relevant effects for the efficient discovery of novel pest control chemicals. The results of insecticidal assays using B. mori larvae suggested that the activation of signal transduction pathways via α-ALOARs might be mainly responsible for the toxicological effects of the heterocycles. [Display omitted] • Effects of heterocycles on α- and β-adrenergic-like octopamine receptors were examined. • The tested compounds displayed structure-dependent receptor subtype selectivity. • The tested compounds showed structure-dependent G protein subtype preference. • The results underscore the need to determine toxicologically relevant effects. [ABSTRACT FROM AUTHOR]

Background: γ-Aminobutyric acid (GABA) receptors (GABARs) are validated targets of insecticides. Bicyclophosphorothionates are a group of insecticidal compounds that act as noncompetitive antagonists of GABARs. We previously reported that the analogs exhibit various degrees of selectivity for housefly versus rat GABARs, depending on substitutions at the 3- and 4-positions. We here sought to elucidate the unsolved mechanisms of the receptor selectivity using quantitative structure-activity relationship (QSAR), molecular docking, and molecular dynamics approaches., Results: Three-dimensional (3D)-QSAR studies using Topomer comparative molecular field analysis quantitatively demonstrated how the introduction of a small alkyl group at the 3-position of bicyclophosphorothionates contributes to the housefly versus rat GABAR selectivity. To investigate the molecular mechanisms of the selective action, bicyclophosphorothionates were docked into housefly Resistance to dieldrin (RDL) GABAR and rat α1β2γ2 GABAR homology models built using the published 3D-structures of human GABARs as templates. The results of molecular docking and molecular dynamics simulations revealed that the 2'Ala and 6'Thr residues of the RDL subunit within the channel are the key amino acids for binding to the housefly GABARs, whereas the 2'Ser residue of γ2 subunit plays a crucial role in binding to rat GABARs., Conclusion: We revealed the molecular mechanisms underlying the selective antagonistic action of bicyclophosphorothionates on housefly versus rat GABARs. The information presented should help design and develop novel, safe GABAR-targeting insecticides. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and ∼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl., (© 2023. The Author(s).)

Background: Fluralaner is the first isoxazoline ectoparasiticide developed to protect companion animals from fleas and ticks. Fluralaner primarily inhibits arthropod γ-aminobutyric acid receptors (GABARs), which are ligand-gated ion channels comprising five subunits arranged around the channel pore. We previously reported that the action site of fluralaner resides at the M1-M3 transmembrane interface between adjacent GABAR subunits. To investigate whether fluralaner interacts with the second transmembrane segment (M2) located deep in the interface, we generated four housefly RDL GABAR mutants with non-conservative amino acid substitutions in the M2 region., Results: Electrophysiological analysis of GABARs expressed in Xenopus oocytes indicated that S313A and S314A mutants exhibited fluralaner sensitivities similar to that of the wild type. M312S mutant exhibited approximately seven-fold lower sensitivity than that of the wild type. Notably, the N316L mutant was almost insensitive to fluralaner., Conclusion: The findings of this study indicate that the conserved external amino acid residues of insect GABAR channels play a critical role in the antagonistic effect of fluralaner. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Dinotefuran, a neonicotinoid, is a unique insecticide owing to its structure and action. We took two approaches that employed insects with controlled expression of nicotinic acetylcholine receptor (nAChR)-encoding genes to gain insight into the uniqueness of dinotefuran. First, we examined the insecticidal activity of dinotefuran and imidacloprid against brown planthoppers (Nilaparvata lugens), in which the expression of eight (of 13) individual subunit-encoding genes was specifically reduced using RNA interference. Knockdown of the tested gene, except one, resulted in a decrease in sensitivity to imidacloprid, whereas the sensitivity of N. lugens to dinotefuran decreased only when two of the eight genes were knocked down. These findings imply that a major dinotefuran-targeted nAChR subtype may contain specific subunits although imidacloprid acts on a broad range of receptor subtypes. Next, we examined the effects of knockout of Drosophila α1 subunit-encoding gene (Dα1) on the insecticidal effects of dinotefuran and imidacloprid. Dα1-deficient flies (Dα1 KO ) demonstrated the same sensitivity to dinotefuran as control flies, but a decreased sensitivity to imidacloprid. This difference was attributed to a reduction in imidacloprid-binding sites in Dα1 KO flies, whereas the binding of dinotefuran remained unchanged. RNA sequencing analysis indicated that Dα2 expression was specifically enhanced in Dα1 KO flies. These findings suggest that changes in Dα1 and Dα2 expression contribute to the differences in the insecticidal activity of dinotefuran and imidacloprid in Dα1 KO flies. Overall, our findings suggest that dinotefuran acts on distinct nAChR subtypes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The ionotropic γ-aminobutyric acid (GABA) receptor (iGABAR) is an important target for insecticides and parasiticides. Our previous studies showed that competitive antagonists (CAs) of insect iGABARs have the potential to be used for developing novel insecticides and that the structural modification of gabazine (a representative CA of mammalian iGABARs) could lead to the identification of novel CAs of insect iGABARs., Results: In the present study, a novel series of 1,3-di- and 1,3,5-trisubstituted 1,6-dihydro-6-iminopyridazines (DIPs) was designed using a versatile strategy and synthesized using facile methods. Electrophysiological studies showed that several target DIPs (30 μM) exhibited excellent antagonistic activities against common cutworm and housefly iGABARs consisting of RDL subunits. The IC 50 values of 3-(4-methoxyphenyl), 3-(4-trifluoromethoxyphenyl), 3-(4-biphenylylphenyl), 3-(2-naphthyl), 3-(3,4-methylenedioxyphenyl), and 3,5-(4-methoxyphenyl) analogs ranged from 2.2 to 24.8 μM. Additionally, several 1,3-disubstituted DIPs, especially 3-(4-trifluoromethoxyphenyl) and 3-(3,4-methylenedioxyphenyl) analogs, exhibited moderate insecticidal activity against common cutworm larvae, with >60% mortality at a concentration of 100 mg kg -1 . Molecular docking studies showed that the oxygen atom on the three-substituted aromatic ring could form a hydrogen bond with Arg254, which may enhance the activity of these DIPs against housefly iGABARs., Conclusion: This systematic study indicated that the presence of a carboxyl side chain shorter by one methylene than that of gabazine at the 1-position of the pyridazine ring is effective for maintaining the stable binding of these DIPs in insect iGABARs. Our study provides important information for the design of novel insect iGABAR CAs. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

γ-Aminobutyric acid receptors (GABARs) mediate fast inhibitory neurotransmission and are targets for insecticides. GABARs are composed of five subunits, the composition of which dictates the pharmacological characteristics of GABARs. Both competitive and noncompetitive GABAR antagonists can be used as insecticides. Gabazine is a potent competitive antagonist of mammalian α1β2γ2 GABARs; however, it is less potent against insect GABARs. To explore how gabazine interacts with GABARs, we examined whether the sensitivity of the small brown planthopper ( Laodelphax striatellus ) RDL GABAR (LsRDLR) to gabazine is increased when its amino acid residues are substituted with α1β2γ2 GABAR residues. In the results, two of the generated mutants showed enhanced gabazine sensitivity. Docking simulations of gabazine using LsRDLR homology models and an α1β2γ2 GABAR cryo-EM structure revealed that the accommodation of gabazine into the "aromatic box" in the orthosteric site lowered the binding energy. This information may help in designing GABAR-targeting insecticides with novel modes of action., (© Pesticide Science Society of Japan 2022. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Ionotropic γ-aminobutyric acid (GABA) receptors (iGABARs) are validated targets of drugs and insecticides. Our previous studies showed that the competitive antagonists of insect iGABARs exhibit insecticidal activities and that the 3-isothiazolol scaffold is used as a lead for developing novel iGABAR antagonists. Here, we designed a novel series of 4-aryl-5-(4-pyridinyl)-3-isothiazolol (4-API) analogs that have various aromatic substituents at the 4-position. Two-electrode voltage clamp experiments showed that all synthesized 4-APIs exhibited antagonistic activity against Musca domestica and Spodoptera litura iGABARs (RDL) expressed in oocytes of Xenopus laevis at 100 μM. Of the 4-APIs, the 4-(1,1'-biphenylyl) analog was the most potent antagonist with IC 50 s of 7.1 and 9.9 μM against M. domestica and S. litura RDL receptors, respectively. This analog also showed a certain insecticidal activity against S. litura larvae, with >75% mortality at 100 μg/g diet. Molecular docking studies with a M. domestica iGABAR model indicated that the π-π stacking interactions formed between the pyridinyl ring and Y252 and between the 4-substituted aromatic group and Y107 might be important for antagonism by the 4-(1,1'-biphenylyl) analog. Our studies provide important information for designing novel iGABAR antagonists and suggest that the 4-APIs acting on iGABARs are promising insecticide leads for further studies.

Background: Root-knot nematodes are plant-parasitic nematodes that cause immense damage to a broad range of cultivated crops by forming root galls, resulting in yield losses in crops. To facilitate the development of faster-acting selective nematicides, we cloned three cDNAs encoding UNC-49B proteins from the southern root-knot nematode Meloidogyne incognita and examined their functional and pharmacological properties by two-electrode voltage clamp electrophysiology using a Xenopus oocyte expression system., Results: The three cloned cDNAs encoded Min-UNC-49B, Min-UNC-49B-L and Min-UNC-49B-XL; the last two proteins have longer N-terminal regions than the first protein. When expressed in Xenopus oocytes, these proteins responded to γ-aminobutyric acid (GABA) to activate currents with high-micromolar or low-millimolar half-maximal effective concentration (EC 50 ) values, indicating the formation of functional homo-pentameric GABA receptors. Fipronil and picrotoxinin inhibited GABA-induced currents with high-nanomolar and low-micromolar half-maximal inhibitory concentration (IC 50 ) values, respectively, in oocytes expressing Min-UNC-49B. The G2'A and T6'M mutations in the second transmembrane domain of Min-UNC-49B enhanced and reduced the sensitivity of Min-UNC-49B to these two antagonists, respectively. Samaderine B and SF-14 inhibited GABA responses in oocytes expressing Min-UNC-49B with low-micromolar and high-nanomolar IC 50 values, respectively. Ivermectin, α-terpineol, thymol and methyl eugenol exerted dual effects on Min-UNC-49B by potentiating currents induced by low concentrations of GABA and inhibiting currents induced by high concentrations of GABA., Conclusion: We have shown that structurally diverse compounds act at Min-UNC-49B GABA receptors. Our results may serve as a starting point to decipher the molecular function of native GABA receptors of plant-parasitic nematodes, which could aid in the structure-based design of novel nematicides. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, β-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Amitraz is a unique formamidine-class acaricide/insecticide that effectively controls ticks, mites, and insect pests. However, the recent emergence of amitraz-resistant cattle ticks is a serious problem that requires an urgent solution. A nonsynonymous single nucleotide polymorphism (A181T) leading to an amino acid substitution (I61F) in the β-adrenergic-like (β-AL) octopamine receptor (OAR) of amitraz-resistant southern cattle ticks (Rhipicephalus microplus) (RmβAOR) was proposed to be a cause of the amitraz resistance. However, it remains unclear whether this substitution exerts any functional effect on the action of amitraz. To make this clear, the functional role of this mutation was examined using an orthologous OAR (BmOAR2) from the silkworm (Bombyx mori)., Results: Both amitraz and its metabolite N 2 -(2,4-dimethylphenyl)-N 1 -methyformamidine (DPMF) elevated intracellular cyclic AMP levels as orthosteric OAR agonists in HEK-293 cells stably expressing BmOAR2. The I45F mutant of BmOAR2 (equivalent to I61F in RmβAOR) was generated and tested for its sensitivity to amitraz and DPMF. The assay result showed that the I45F mutation reduces the potency of DPMF to a level similar to that of the endogenous agonist (R)-OA in wild-type BmOAR2., Conclusion: The amino acid substitution found in the first transmembrane segment of RmβAOR most likely causes target-site insensitivity to DPMF, which might contribute to the resistance of R. microplus to amitraz. This needs to be further confirmed using RmβAOR. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Dinotefuran (DTF) is a systemic neonicotinoid insecticide characterized by a tetrahydrofuran ring. In the present study, we examined the characteristics of DTF binding to native nicotinic acetylcholine receptors (nAChRs) expressed in the American cockroach Periplaneta americana using radioligand-binding methods. The Scatchard analysis, using [ 3 H]imidacloprid (IMI), indicated that IMI has a single class of high-affinity binding sites in the P. americana nerve cord. In contrast, the Scatchard analysis using [ 3 H]DTF indicated that DTF has two different classes of binding sites. Both DTF and IMI were found to bind to one of the classes, for which DTF showed low affinity. The other class, for which DTF showed high affinity, was localized in the abdominal nerve cord but not in the thoracic nerve cord. IMI showed low affinity for the high-affinity DTF binding sites. Our data suggest that DTF binds with high affinity to a nAChR subtype distinct from the high-affinity subtype for IMI. This difference might be responsible, at least in part, for the difference in resistance development to DTF and IMI in P. americana., (Copyright © 2020 Elsevier Inc. All rights reserved.)