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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 domesticaand Spodoptera lituraiGABARs (RDL) expressed in oocytes of Xenopus laevisat 100 μM. Of the 4-APIs, the 4-(1,1′-biphenylyl) analog was the most potent antagonist with IC50s of 7.1 and 9.9 μM against M. domesticaand S. lituraRDL receptors, respectively. This analog also showed a certain insecticidal activity against S. lituralarvae, with >75% mortality at 100 μg/g diet. Molecular docking studies with a M. domesticaiGABAR 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.

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.Graphical AbstractLigand-gated chloride channels and octopamine receptors are validated invertebrate pest control targets with multiple potential sites for new ligands.

Ionotropic γ-aminobutyric acid (GABA) receptors (GABARs) represent an important insecticide target. Currently used GABAR-targeting insecticides are non-competitive antagonists (NCAs) of these receptors. Recent studies have demonstrated that competitive antagonists (CAs) of GABARs have functions of inhibiting insect GABARs similar to NCAs and that they also exhibit insecticidal activity. CAs have different binding sites and different mechanisms of action compared to those of NCAs. Therefore, GABAR CAs should have the potential to be developed into novel insecticides, which could be used to overcome the developed resistance of insect pests to conventional NCA insecticides. Although research on insect GABAR CAs has lagged behind that on mammalian GABAR CAs, research on the CAs of insect ionotropic GABARs has made great progress in recent years, and several series of heterocyclic compounds, such as 3-isoxazolols and 6-iminopyridazines, have been identified as insect GABAR CAs. In this review, we briefly summarize the design strategies, structures, and biological activities of the novel GABAR CAs that have been found in the past decade. Updated information about GABAR CAs may benefit the design and development of novel GABAR-targeting insecticides.

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly (Musca domestica) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of MuscaGABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in MuscaGluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species.

Abstract: A series of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-1,2,3-triazole analogues have been tested for antimicrobial activity against thirteen bacteria and three fungi in order to observe the broad spectrum activity. Among the compounds tested, the C-4-chloropropyl derivative exhibited potent antibacterial and antifungal activities. The C-4-substituted regioisomers of 1-(2,6-dichloro-4-trifluoromethylphenyl)-1H-1,2,3-triazole exhibited moderate selectivity to broad spectrum activity, compared to that of 5-substituted regioisomers.

4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl− channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH.

Acyclic noncompetitive antagonists of ionotropic γ-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50of 88 nM. The potency conferred by the p-substituent decreased in the order CC(CH2)2COOH>CC(CH2)2COOCH3>CCH>Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less potent. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50of 0.54 μM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.

To investigate the role of the heterocyclic moieties of nitrogen-containing phenyl heterocyclic compounds (PHCs) in interaction with γ-aminobutyric acid (GABA)-gated chloride channels, diverse classes of PHCs were examined for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive GABA antagonist, to housefly head and rat brain membranes. PHCs that inhibited [3H]EBOB binding include pyrazoles; 1H-1,2,3-triazoles; a 1H-1,2,4-triazole; 1,3,4-oxadiazol-2(3H)-ones; a 1,3,4-oxadiazole-2(3H)-thione; 1,2,4-oxadiazoles; a 1,2,4-thiadiazole; thiazoles; a 4(3H)-pyrimidinone; and a 2,4(1H,3H)-pyrimidinedione. An analogue (1) of the pyrazole insecticide fipronil, bearing an SCF3group in place of the S(O)CF3, was found to be the most potent inhibitor with IC50sof 7.55 and 177 nM in housefly head and rat brain membranes, respectively. 3-(2,6-Dichloro-4-trifluoromethyl phenyl)-5-t-butyl-1,3,4-oxadiazol-2(3H)-one exhibited the highest selectivity for housefly GABA receptors versus rat receptors (IC50rat/IC50fly >204). PHCs that exhibited a comparable selectivity include a pyrazole and a 1H-1,2,3-triazole. Interaction of 16 selected PHCs with rat brain GABA-gated channels was also examined using [3S]t-butylbicyclophosphorothionate (TBPS), a radioligand for the mammalian noncompetitive antagonist site. A plot of pIC50sof 12 PHCs revealed a close correlation (r= 0.93) between their potency in inhibiting [3H]EBOB and [35S]TBPS binding. Scatchard analyses of the inhibition of [35S]TBPS binding by 1suggested a competitive-type inhibition. A plot of the potency of 14 PHCs in inhibiting [3H]EBOB binding to housefly head membranes against their piperonyl butoxide-synergized insecticidal effect on German cockroaches yielded a close correlation (r= 0.89), with the exception of five triazoles and a 2,4(1H,3H)-pyrimidinedione. Although several selected PHCs also inhibited the specific binding of [3H]batrachotoxinin A 20-α-benzoate, a tritiated analogue of the sodium channel activator batrachotoxinin, to synaptosomes and membranes prepared from mouse brains, housefly heads, and American cockroach nerve cords, the concentrations required were higher than those of standard compounds producing significant effects on this system. The results demonstrate that a variety of five- and six- membered, nitrogen-containing heterocyclic compounds, bearing a 2,6-dichloro-4-trifluoromethyphenyl or 2,4,6- trichlorophenyl group, interact with ionotropic GABA receptors. Several PHCs display higher affinities for housefly GABA receptors and high selectivity as compared to rat GABA receptors. PHCs' insecticidal activity is mediated by their interaction with GABA-gated chloride channels.

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [³H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C3 length and the 4-alkyl groups of C4 length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC50 = 45.2 nM), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC50 = 62.2 nM). Their receptor selectivities (rat IC50/housefly IC50) were 52 and 0.038, respectively. The insecticidal activity (LD50) of 20 active analogues was well correlated with their potency (IC50) in inhibiting [³H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABAA receptors. © 1999 Society of Chemical Industry

Anisatin, a toxic, insecticidally active component of Sikimi plant, is known to act on the GABA system. In order to elucidate the mechanism of anisatin interaction with the GABA system, whole‐cell and single‐channel patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture.Repeated co‐applications of GABA and anisatin suppressed GABA‐induced whole‐cell currents with an EC50of 1.10 μM. No recovery of currents was observed after washout with anisatin‐free solution.However, pre‐application of anisatin through the bath had no effect on GABA‐induced currents. The decay phase of currents was accelerated by anisatin. These results indicate that anisatin suppression of GABA‐induced currents requires opening of the channels and is use‐dependent.Anisatin suppression of GABA‐induced currents was not voltage dependent.Picrotoxinin attenuated anisatin suppression of GABA‐induced currents. [3H]‐EBOB binding to rat brain membranes was competitively inhibited by anisatin. These data indicated that anisatin bound to the picrotoxinin site.At the single‐channel level, anisatin did not alter the open time but prolonged the closed time. The burst duration was reduced and channel openings per burst were decreased indicating that anisatin decreased the probability of openings.

Twenty-eight picrotoxane terpenoids, including picrodendrins isolated from the Euphorbiaceae plant, Picrodendron baccatum(L.) Krug and Urban, have been evaluated for their ability to inhibit the specific binding of [3H]EBOB, the noncompetitive antagonist of ionotropic GABA receptors, to rat-brain and housefly (Musca domesticaL.)-head membranes. Picrodendrin Q was the most potent competitive inhibitor of [3H]EBOB binding, with IC50values of 16nM (rat) and 22nM (Musca). We find that the spiro γ-butyrolactone moiety at the 13-position, which contains a carbonyl group conjugated with an unsaturated bond, and the substituents at the 4-position play important roles in the interaction of picrodendrins with their binding site in rat receptors. In contrast, such structural features are not strictly required in the case of the interaction with Muscareceptors; the spiro saturated γ-butyrolactone moiety at the 13-position, which bears the 16-sp3carbon atom, and the hydroxyl groups at various positions are somewhat tolerated. Quantitative structure-activity studies have clearly shown that the electronegativity of the 16-carbon atom and the presence or absence of the 4- and 8-hydroxyl groups are important determinants of the potency of nor-diterpenes in Muscareceptors, while the negative charge on the 17-carbonyl oxygen atom is likely important in the case of rat receptors. These findings indicate that there are significant differences between the structures of the complementary binding sites in rat GABA receptors and MuscaGABA receptors. We also infer differences between native MuscaGABA receptors and the Drosophila Rdlsubunit-containing homo-oligomeric GABA receptors in the structures of their binding sites.

Quantitative structure–activity relationships for insecticidal activity (against houseflies) and competitive activity against a specific [³⁵S]tert-butylbicyclophosphorothionate binding (to rat brain membranes) of some picrotoxinin-type 4-aminobutyric acid antagonists, including γ-BHC, endosulfan, bicyclophosphates, dioxatricyclododecenes and related compounds, were examined three-dimensionally using comparative molecular field analysis (CoMFA). The antagonists were classified into two series according to their molecular shapes: i.e. whether their structure was ‘linearly’ extended beyond the ‘mast-head’ position of the ‘boat-like’ skeletons (series 1) or not (series 2). The CoMFA showed that the slopes in steric and electrostatic fields around the molecule were significant for both series in governing the potency variations in insecticidal and binding activities. Hydrophobicity, a possible factor controlling transport behaviour of compounds, was significant in governing variations in insecticidal activity, but not for the case of the rat membrane binding. Assuming that there is a slight topological difference between series 1 and 2 compounds in terms of the mode of binding with the housefly receptor site, the insecticidal activity was analysable with a single equation for the combined set of compounds, but the rat membrane binding was not. The sterically and electrostatically favourable regions surrounding the molecular series indicated by CoMFA were roughly located at positions so as to interact with the binding subsites on the receptors proposed previously. © of SCI.

Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.

Different structural features govern the interaction of picrodendrins and related terpenoids with rat and with housefly GABA receptors. This supports previous studies which suggest that there are significant differences between the structures of the binding sites in these two receptors.