The mode of action of isocycloseram: A novel isoxazoline insecticide.

Isocycloseram is a novel isoxazoline insecticide and acaricide with activity against lepidopteran, hemipteran, coleopteran, thysanopteran and dipteran pest species. Isocycloseram selectively targets the invertebrate Rdl GABA receptor at a site that is distinct to fiproles and organochlorines. The widely distributed cyclodiene resistance mutation, A301S, does not affect sensitivity to isocycloseram, either in vitro or in vivo, demonstrating the suitability of isocylsoseram to control pest infestations with this resistance mechanism. Detailed studies demonstrated that the binding sites relevant to the insecticidal activity of avermectins and isocycloseram are distinct. Isocycloseram was shown to compete for binding with metadiamide insecticides related to broflanilide. In addition, a G335M mutation in the third transmembrane domain of the Rdl GABA receptor, impaired the ability of both isocycloseram and metadiamides to block the GABA mediated response. As such the Insecticides Resistance Action Committee (IRAC) has classified isocycloseram in Group 30 "GABA-Gated Chloride Channel Allosteric Modulators".

Insecticides, biologics and nematicides: Updates to IRAC's mode of action classification - a tool for resistance management.

Insecticide resistance has been and continues to be a significant problem for invertebrate pest control. As such, effective insecticide resistance management (IRM) is critical to maintain the efficacy of current and future insecticides. A technical group within CropLife International, the Insecticide Resistance Action Committee (IRAC) was established 35 years ago (1984) as an international association of crop protection companies that today spans the globe. IRAC's focus is on preserving the long-term utility of insect, mite, and most recently nematode control products through effective resistance management to promote sustainable agriculture and improved public health. A central task of IRAC has been the continual development and documentation of the Mode of Action (MoA) Classification scheme, which serves as an important tool for implementing IRM strategies focused on compound rotation / alternations. Updates to the IRAC MoA Classification scheme provide the latest information on the MoA of current and new insecticides and acaricides, and now includes information on biologics and nematicides. Details for these new changes and additions are reviewed herein.

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel composed of 5 protein subunits arranged around a central cation selective pore. Several classes of natural and synthetic insecticides mediate their effect through interacting at nAChRs. This review examines the basic pharmacology of the neonicotinoids and related chemistry, with an emphasis on sap-feeding insects from the order Hemiptera, the principle pest target for such insecticides. Although the receptor subunit stoichiometry for endogenous invertebrate nAChRs is unknown, there is clear evidence for the existence of distinct neonicotinoid binding sites in native insect preparations, which reflects the predicted wide repertoire of nAChRs and differing pharmacology within this insecticide class. The spinosyns are principally used to control chewing pests such as Lepidoptera, whilst nereistoxin analogues are used on pests of rice and vegetables through contact and systemic action, the pharmacology of both these insecticides is unique and different to that of the neonicotinoids.

Target-site resistance to neonicotinoids.

Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

Investigating nicotinic acetylcholine receptor expression in neonicotinoid resistant Myzus persicae FRC.

The peach-potato aphid Myzus persicae is a pest of many commercial crops due to its polyphagous nature of feeding and has a well-documented history of acquiring resistance to insecticides. In 2009 a strain (M. persicae FRC) emerged in southern France with a point mutation (R81T) at the nicotinic acetylcholine receptor (nAChR), the target site for neonicotinoids such as imidacloprid. This point mutation was associated with the loss of the high affinity imidacloprid binding site (pM Kd), with the single remaining binding site (low nM Kd) highly overexpressed compared to laboratory controls (Bass et al., 2011 [1]). Here we report that after 2years of continuous selection in the glass house environment with neonicotinoids, the total level of IMD-sensitive nAChRs (low nM Kd) in M. persicae FRC is now comparable to laboratory controls (pM and low nM Kd). Interestingly, despite this large reduction in IMD-sensitive nAChRs, this was not associated with any significant alteration in NNIC-lethality. Additionally, sustained absence of neonicotinoid-selection did not alter nAChR protein levels. We suggest that alterations in nAChR protein expression level described in the original characterisation of the field-isolated M. persicae FRC is unlikely to have been a direct consequence of the R81T mutation. Rather, we speculate that nAChR expression in aphids is likely influenced by as yet unknown conditions in the natural field environment that are absent in the laboratory setting.

Investigating the mode of action of sulfoxaflor: a fourth-generation neonicotinoid.

BACKGROUND: The precise mode of action of sulfoxaflor, a new nicotinic acetylcholine receptor-modulating insecticide, is unclear. A detailed understanding of the mode of action, especially in relation to the neonicotinoids, is essential for recommending effective pest management practices. RESULTS: Radiolabel binding experiments using a tritiated analogue of sulfoxaflor ([(3) H]-methyl-SFX) performed on membranes from Myzus persicae demonstrate that sulfoxaflor interacts specifically with the high-affinity imidacloprid binding site present in a subpopulation of the total nAChR pool. In competition studies, imidacloprid-like neonicotinoids displace [(3) H]-methyl-SFX at pM concentrations. The effects of sulfoxaflor on the exposed aphid nervous system in situ are analogous to those of imidacloprid and nitenpyram, and finally the high-affinity sulfoxaflor binding site is absent in a Myzus persicae strain (clone FRC) possessing a single amino acid point mutation (R81T) in the ß-nAChR, a region critical for neonicotinoid interaction. CONCLUSION: The nicotinic acetylcholine receptor pharmacological profile of sulfoxaflor in aphids is consistent with that of imidacloprid. Additionally, the insecticidal activity of sulfoxaflor and the current commercialised neonicotinoids is affected by the point mutation in FRC Myzus persicae. Therefore, it is suggested that sulfoxalfor be considered a neonicotinoid, and that this be taken into account when recommending insecticide rotation partnering for effective resistance management programmes.

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

Novel diamide insecticides: sulfoximines, sulfonimidamides and other new sulfonimidoyl derivatives.

Novel insecticidal anthranilamides with elaborated sulfur-containing groups are described. The synthesis of compounds with functional groups such as sulfoximines and scarcely reported groups such as sulfonimidoyl hydrazides and hydroxylamides, their in vitro and in vivo biological activity as well as their physical properties are reported.

Mutation of a nicotinic acetylcholine receptor ß subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.

BACKGROUND: Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. RESULTS: Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid) to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR). Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpß1) genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR ß1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T). CONCLUSION: Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also provides further validation of exisiting models of neonicotinoid binding and selectivity for insect nAChRs.

Higher-order organization and regulation of adenylyl cyclases.

There is increasing awareness of the compartmentalization of cAMP signalling--the means by which cAMP levels change in discrete domains of the cell with discrete local consequences. Current developments in understanding the organization of adenylyl cyclases in the plasma membrane are illuminating how the earliest part of cAMP compartmentalization could occur. This review focuses on recent findings regarding three levels of adenylyl cyclase organization--oligomerization, positioning to lipid rafts and participation in multiprotein signalling complexes. This organization, coupled with the role of scaffolding proteins in arranging the downstream effectors of cAMP, helps to identify complexes that greatly facilitate the translation of enzyme activation into local consequences.

A direct interaction between the N terminus of adenylyl cyclase AC8 and the catalytic subunit of protein phosphatase 2A.

Although protein scaffolding complexes compartmentalize protein kinase A (PKA) and phosphodiesterases to optimize cAMP signaling, adenylyl cyclases, the sources of cAMP, have been implicated in very few direct protein interactions. The N termini of adenylyl cyclases are highly divergent, which hints at isoform-specific interactions. Indeed, the Ca(2+)-sensitive adenylyl cyclase 8 (AC8) contains a Ca(2+)/calmodulin binding site on the N terminus that is essential for stimulation of activity by the capacitative entry of Ca(2+) in the intact cell. Here, we have used the N terminus of AC8 as a bait in a yeast two-hybrid screen of a human embryonic kidney (HEK) 293 cell cDNA library and identified the catalytic subunit of the serine/threonine protein phosphatase 2A (PP2A(C)) as a binding partner. Confirming the highly specific nature of this novel interaction, glutathione-S-transferase fusion proteins containing the full-length N terminus of AC8 affinity precipitated catalytically active PP2A(C) from both HEK293 and mouse forebrain membranes-the latter a normal source of AC8. The scaffolding subunit of PP2A (PP2A(A); 65 kDa) was also precipitated by the N terminus of AC8, indicating that AC8 may occur in a complex with the PP2A core dimer. The interaction between the N terminus of AC8 and PP2A(C) was antagonized by Ca(2+)/calmodulin. However, PP2A(C) and Ca(2+)/calmodulin did not share identical binding specificities in the N terminus of AC8. PKA-mediated phosphorylation did not influence either calmodulin or PP2A(C) association with AC8. In addition, both PP2A(C) and AC8 occurred in lipid rafts. These findings are the first demonstration of an association between adenylyl cyclase and any downstream element of cAMP signaling.

Localized Na+/H+ exchanger 1 expression protects Ca2+-regulated adenylyl cyclases from changes in intracellular pH.

The Ca2+-sensitive adenylyl cyclases (ACs) are exclusively regulated by capacitative Ca2+ entry (CCE) in nonexcitable cells. The present study investigates whether this Ca2+-dependent modulation of AC activity is further regulated by local pH changes that can arise beneath the plasma membrane as a consequence of cellular activity. Ca2+ stimulation of AC8 expressed in HEK 293 cells and inhibition of endogenous AC6 in C6-2B glioma cells exhibited clear sensitivity to modest pH changes in vitro. Acid pH (pH 7.14) reduced the Ca2+ sensitivity of both ACs, whereas alkaline pH (pH 7.85) enhanced the responsiveness of the enzymes to Ca2+, compared with controls (pH 7.50). Surprisingly, in the intact cell, the response of AC8 and AC6 to CCE was largely unperturbed by similar changes in intracellular pH (pH(i)), imposed using a weak acid (propionate) or weak base (trimethylamine). A range of hypotheses were tested to identify the mechanism(s) that could underlie this lack of pH effect in the intact cell. The pH sensitivity of CCE in HEK 293 cells is likely to dampen the effects of pH(i) on Ca2+-regulated ACs and may partly explain the discrepancy between in vitro and in vivo data. However, we have found that the Na+/H+ exchanger (NHE), NHE1, is functionally active in these cells, and like AC8 (and AC6) it resides in lipid rafts or caveolae, which may create cellular microdomains where pH(i) is tightly regulated. An abundance of NHE1 in these cellular subdomains may generate a privileged environment that protects the Ca2+-sensitive ACs and other caveolar proteins from local acid shifts.

The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts.

Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress.

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.

Phosphatidylinositol 3-kinase is a central mediator of NMDA receptor signalling to MAP kinase (Erk1/2), Akt/PKB and CREB in striatal neurones.

Ca2+ influx through NMDA receptors can initiate molecular changes in neurones which may underlie synaptic plasticity, neuronal development, survival and excitotoxicity. Signalling through the MAP kinase (Erk1/2) cascade may be central to these processes. We previously demonstrated that Ca2+-permeable AMPA receptors activate Erkl/2 through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent mechanism. We now report that NMDA receptor activation of Erk1/2 was also blocked by inhibitors of PI 3-kinase (LY 294002, wortmannin). In addition, pre-treatment of neurones with pertussis toxin inhibited NMDA-induced Erk1/2 activation, indicating a role for heterotrimeric Gi/o proteins. PI 3-kinase directs activation of the serine-threonine kinase Akt (PKB). Treatment of striatal neurones with glutamate induced a rapid Ca2+-dependent and PI 3-kinase-dependent phosphorylation of Akt (Ser473), which was not blocked by the Mek inhibitors PD98059 or U0126. Targets for Erk1/2 and Akt pathways include transcription factors. Glutamate-induced phosphorylation of cAMP response element binding protein (CREB; Ser133) was partially blocked with either PD98059, U0126, LY294002 or wortmannin but was very strongly inhibited on co-application of LY294002 and PD98059. We propose that NMDA receptor stimulation can activate Erk1/2 and Akt signalling pathways in a PI 3-kinase dependent manner which may target CREB in the nucleus.

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca(2+) and PI3-kinase.

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.