New and selective ryanodine receptor activators for insect control

Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety. Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera. This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle. The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors.     

杀虫药剂的神经毒理学研究进展

大多数杀虫药剂都具有较强的神经毒性,它们对神经系统的作用靶标不同。有机磷类杀虫剂不仅抑制乙酰胆碱酯酶活性和乙酰胆碱受体功能,影响乙酰胆碱的释放,而且还具有非胆碱能毒性,有些有机磷杀虫剂还能引发迟发性神经毒性。新烟碱类杀虫剂作为烟碱型乙酰胆碱受体（nAChR）的激动剂,作用于该类受体的α亚基;它对昆虫的毒性比对哺乳动物的毒性大得多,乃是因为它对昆虫和哺乳动物nAChR的作用位点不同。拟除虫菊酯类杀虫剂主要作用于神经细胞钠通道,引起持续开放,导致传导阻滞;该类杀虫剂也可抑制钙通道。另外,这类杀虫剂还干扰谷氨酸递质和多巴胺神经元递质的释放。拟除虫菊酯类杀虫剂对昆虫的选择毒性很可能是因为昆虫神经元的钠通道结构与哺乳动物的不同。阿维菌素类杀虫剂主要作用于γ-氨基丁酸（GABA）受体,它能促进GABA的释放,增强GABA与GABA受体的结合,使氯离子内流增加,导致突触后膜超级化。由于这类杀虫剂难以穿透脊椎动物的血脑屏障而与中枢神经系统的GABA受体结合,故该类杀虫剂对脊椎动物的毒性远低于对昆虫的毒性。多杀菌素类杀虫剂可与中枢神经系统的nAChR作用,引起Ach长时间释放,此外,这类杀虫剂还可作用于昆虫的GABA受体,改变GABA门控氯通道的功能。     

褐飞虱对吡虫啉的抗性机理和靶标分子毒理学

褐飞虱Nilaparvata lugens是水稻最重要的害虫之一,长期依赖化学防治导致了该害虫对不同类型杀虫剂抗性的产生,对新烟碱类杀虫剂吡虫啉高水平抗性的产生更是造成了巨大的粮食生产损失。近年来在褐飞虱对吡虫啉抗性机理,以及在抗药性机理研究推动下吡虫啉作用靶标褐飞虱神经系统烟碱型乙酰胆碱受体（nicotinic acetylcholine receptors, nAChRs）毒理学等方面取得了许多研究进展。nAChRs是昆虫神经系统中最重要的神经递质受体,是几类重要杀虫剂的作用靶标,其中以新烟碱类杀虫剂为代表。通过对比敏感品系和室内连续筛选获得的高抗吡虫啉品系,在褐飞虱两个nAChRs亚基Nlα1和Nlα3中均发现了抗性相关点突变Y151S,该突变导致了受体与吡虫啉结合亲和力的显著下降,而对内源神经递质乙酰胆碱的亲和力影响很小。Nlα1与褐飞虱另外两个亚基Nlα2和Nlβ1共聚成一个受体,构成吡虫啉低亲和力结合位点;Nlα3与褐飞虱另外两个亚基Nlα8和Nlβ1共聚成一个受体,构成吡虫啉高亲和力结合位点。不仅褐飞虱nAChRs与吡虫啉抗性相关,某些nAChRs附属蛋白也直接影响褐飞虱对吡虫啉的抗性,如Lynx蛋白。关于褐飞虱nAChRs组成、抗药性相关变异、受体附属蛋白对抗药性的影响等方面的研究,均为国内外前沿报道,不仅有助于对新烟碱类杀虫剂抗性机理的理解,对昆虫nAChRs毒理学同样具有很大的推动作用。     

Novel nicotinic action of the sulfoximine insecticide sulfoxaflor

The novel sulfoximine insecticide sulfoxaflor is as potent or more effective than the neonicotinoids for toxicity to green peach aphids (GPA, Myzus persicae). The action of sulfoxaflor was characterized at insect nicotinic acetylcholine receptors (nAChRs) using electrophysiological and radioligand binding techniques. When tested for agonist properties on Drosophila melanogaster D??2 nAChR subunit co-expressed in Xenopus laevis oocytes with the chicken ??2 subunit, sulfoxaflor elicited very high amplitude (efficacy) currents. Sulfoximine analogs of sulfoxaflor were also agonists on D??2/??2 nAChRs, but none produced maximal currents equivalent to sulfoxaflor nor were any as toxic to GPAs. Additionally, except for clothianidin, none of the neonicotinoids produced maximal currents as large as those produced by sulfoxaflor. These data suggest that the potent insecticidal activity of sulfoxaflor may be due to its very high efficacy at nAChRs. In contrast, sulfoxaflor displaced [³H]imidacloprid (IMI) from GPA nAChR membrane preparations with weak affinity compared to most of the neonicotinoids examined. The nature of the interaction of sulfoxaflor with nAChRs apparently differs from that of IMI and other neonicotinoids, and when coupled with other known characteristics (novel chemical structure, lack of cross-resistance, and metabolic stability), indicate that sulfoxaflor represents a significant new insecticide option for the control of sap-feeding insects.     

Functional co-expression of two insect nicotinic receptor subunits (Nlα3 and Nlα8) reveals the effects of a resistance-associated mutation (Nlα3Y151S) on neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. Previously, we have identified a nAChR point mutation (Y151S) associated with insecticide resistance in the brown planthopper Nilaparvata lugens . Although this mutation has been identified in two different N. lugens nAChR subunits (Nlα1 and Nlα3) because of difficulties in heterologous expression of Nlα3; its influence on agonist potency has been examined only in Nlα1-containing nAChRs. Here we describe the cloning of a novel nAChR subunit from N. lugens (Nlα8), together with evidence for its co-assembly with Nlα3 in native and recombinant nAChRs. This has, for the first time, enabled the functional effects of the Nlα3^Y151S mutation to be examined. The Nlα3^Y151S mutation has little effect on agonist potency of acetylcholine but has a dramatic effect on neonicotinoid insecticides (reducing I _max values and increasing EC₅₀ values). The apparent affinity of neonicotinoids was higher and the effect of the Y151S mutation on neonicotinoid agonist potency was more profound in Nlα3-containing, rather than Nlα1-containing nAChR. We conclude that Nlα3- and Nlα1-containing nAChRs may be representative of two distinct insect nAChR populations.     

Blockage of chloride channels and anion transporters with pesticidal natural products and their synthetic analogs

Ligand-gated chloride channels mediate a variety of functions in excitable membranes of nerve and muscle in insects, and have a long history as targets for neurotoxic insecticides. Recent findings from our laboratory confirm that the natural product silphinenes and their semi-synthetic analogs share a mode of action with the established ligand-gated chloride channel antagonist, picrotoxinin. The silphinenes are non-selective, being roughly equipotent on insect and mammalian receptors, but also possess lethal and neurotoxic effects on a dieldrin-resistant strain of Drosophila melanogaster. These findings suggest that silphinenes act on insect GABA receptors in a way that is different from picrotoxinin, and it is possible that resistant insect populations in the field could be controlled with insecticidal compounds derived from the silphinenes. Voltage-gated chloride channels and anion transporters provide additional classes of validated targets for insecticidal/nematicidal action. Anion transporter blockers are toxic to insects via an action on the gut, and RNAi studies implicate voltage-gated chloride channels in nematode muscle as another possible target. There was no cross resistance to DIDS in a dieldrin-resistant strain of Drosophila melanogaster, and no evidence for neurotoxicity. The potent paralytic actions of anion transporter blockers against nematodes, and stomach poisoning activity against lepidopteran larvae suggests they are worthy of further investigation as commercial insecticidal/nematicidal agents.     

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC(50) value for ACh of 19 microm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 microm ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.     

A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoid insecticides

Neonicotinoid insecticides are potent selective agonists of insect nicotinic acetylcholine receptors (nAChRs). Since their introduction in 1991, resistance to neonicotinoids has been slow to develop, but it is now established in some insect field populations such as the planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. We have reported recently the identification of a target-site mutation (Y151S) within two nAChR subunits (Nlalpha1 and Nlalpha3) from a laboratory-selected field population of N. lugens. In the present study, we have examined the influence of this mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes (as hybrid nAChRs, co-expressed with a rat beta2 subunit). The agonist potency of several nicotinic agonists has been examined, including all of the neonicotinoid insecticides that are currently licensed for either crop protection or animal health applications (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). The Y151S mutation was found to have no significant effect on the maximal current (I(max)) observed with the endogenous agonist, acetylcholine. In contrast, a significant reduction in I(max) was observed for all neonicotinoids (the I(max) for mutant nAChRs ranged from 13 to 81% of that observed on wild-type receptors). In addition, nAChRs containing the Y151S mutation caused a significant rightward shift in agonist dose-response curves for all neonicotinoids, but of varying magnitude (shifts in EC(50) values ranged from 1.3 to 3.6-fold). The relationship between neonicotinoid structure and their potency on nAChRs containing the Y151S target-site mutation is discussed.     

Heteromeric co-assembly of two insect nicotinic acetylcholine receptor α subunits: influence on sensitivity to neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlα1 and Nlα2 cloned from the brown planthopper Nilaparvata  lugens , a major insect pest of rice crops in many parts of Asia. The influence of Nlα1 and Nlα2 subunits upon the functional properties of recombinant nAChRs has been examined by expression in Xenopus oocytes. In addition, the influence of a Nlα1 mutation (Y151S), which has been linked to neonicotinoid lab generated resistance in N. lugens , has been examined. As in previous studies of insect α subunits, functional expression has been achieved by co-expression with the mammalian β2 subunit. This approach has revealed a significantly higher apparent affinity of imidacloprid for Nlα1/β2 than for Nlα2/β2 nAChRs. In addition, evidence has been obtained for the co-assembly of Nlα1 and Nlα2 subunits into 'triplet' nAChRs of subunit composition Nlα1/Nlα2/β2. Evidence has also been obtained which demonstrates that the resistance-associated Y151S mutation has a significantly reduced effect on neonicotinoid agonist activity when Nlα1 is co-assembled with Nlα2 than when expressed as the sole α subunit in a heteromeric nAChR. These findings may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance in insect field populations.     

Synthesis of some ester derivatives of 4′-demethoxyepipodophyllotoxin/2′-chloro-4′-demethoxyepipodophyllotoxin as insecticidal agents against oriental armyworm,Mythimna separata Walker

Podophyllotoxin is a naturally occurring non-alkaloid toxin isolated from the roots and rhizomes of Podophyllum peltatum and P. hexandrum. In continuation of our program aimed at the discovery and development of natural product-based insecticides, two series of ester derivatives of 4′-demethoxyepipodophyllotoxin/2′-chloro-4′-demethoxyepipodophyllotoxin were prepared. The structures of the target compounds were well characterized by 1H NMR, IR, optical rotation and mp. The precise three-dimensional structural information of 8j was further determined by single-crystal X-ray diffraction. Their insecticidal activity was tested against Mythimna separata Walker. These compounds showed delayed insecticidal activity. Among all derivatives, some compounds showed more potent insecticidal activity than toosendanin against M. separata; especially compounds 8k and 9k exhibited the most potent activity with the final mortality rates of 71.4%. Their structure–activity relationships were discussed.     

Insect ryanodine receptors: molecular targets for novel pest control chemicals

Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. They are located on the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons and many other cell types. Ryanodine, a plant alkaloid and an important ligand used to characterize and purify the receptor, has served as a natural botanical insecticide, but attempts to generate synthetic commercial analogues of ryanodine have proved unsuccessful. Recently two classes of synthetic chemicals have emerged resulting in commercial insecticides that target insect RyRs. The phthalic acid diamide class has yielded flubendiamide, the first synthetic ryanodine receptor insecticide to be commercialized. Shortly after the discovery of the phthalic diamides, the anthranilic diamides were discovered. This class has produced the insecticides Rynaxypyr(R) and Cyazypyrtrade mark. Here we review the structure and functions of insect RyRs and address the modes of action of phthalic acid diamides and anthranilic diamides on insect ryanodine receptors. Particularly intersting is the inherent selectivity both chemical classes exhibit for insect RyRs over their mammalian counterparts. The future prospects for RyRs as a commercially-validated target site for insect control chemicals are also considered.     

The mode of action of a nitroconjugated neonicotinoid and the effects of target site mutation Y151S on its potency

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) with -NO₂ or -CN group in trans-configuration. Previously we reported the excellent insecticidal activity of a series of nitroconjugated neonicotinoids with -NO₂ or -CN group in cis-configuration by replacing nitromethylene pharmacophore with a nitroconjugated system. To understand the action mode of these nitroconjugated neonicotinoids, a representative member IPPA152201 was chosen to perform toxicity and pharmacology studies. IPPA152201 showed a comparable toxicity with imidacloprid against Nilaparvata lugens in a susceptible strain and had no significant cross-resistance in an imidacloprid resistant strain. IPPA152201 showed good efficacies on the isolated cockroach neurons (pEC₅₀ = 5.91 ± 0.14) and the evoked responses by IPPA152201 could be blocked by the typical nAChRs antagonists methyllycaconitine citrate (MLA) and dihydro-??-erythroidine (DH??E), with pIC₅₀ of 6.56 ± 0.07 and 6.89 ± 0.12. The efficacy of IPPA152201 on hybrid receptors Nl??1/??2 in Xenopus oocytes and response inhibition by MLA and DH??E were also observed. These data demonstrate that IPPA152201 acts on insect nAChRs as an agonist. In addition, the influence of a Nl??1 mutation (Y151S), which has been linked to the lab-generated neonicotinoid resistance in N. lugens, has been examined. Compared to the wildtype Nl??1/??2, this mutation reduced I_max for IPPA152201 to 63.2% and caused a 1.5-fold increase in EC₅₀, which is much smaller than the effects on imidacloprid. The high insecticidal activity and little influence by Y151S mutation make IPPA152201 to be a potential insecticide to manage N. lugens.     

Cytochromes P450 and insecticide resistance.

The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects, and the different techniques that have been used to isolate individual insect P450s. Next, insecticide resistance is briefly described, and then historical information about monooxygenase-mediated insecticide resistance is reviewed. For any case of monooxygenase-mediated resistance, identification of the P450(s) involved, out of the dozens that are present in an insect, has proven very challenging. Therefore, the next section of the review focuses on the minimal criteria for establishing that a P450 is involved in resistance. This is followed by a comprehensive examination of the literature concerning the individual P450s that have been isolated from insecticide resistant strains. In each case, the history of the strain and the evidence for monooxygenase-mediated resistance are reviewed. The isolation and characterization of the P450(s) from the strain are then described, and the evidence of whether or not the isolated P450(s) is involved in resistance is summarized. The remainder of the review summarizes our current knowledge of the molecular basis of monooxygenase-mediated resistance and the implications for the future. The importance of these studies for development of effective insecticide resistance management strategies is discussed.     

Neonicotinic analogues: Selective antagonists for α4β2 nicotinic acetylcholine receptors

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4β2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4β2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-¹²⁵I]bungarotoxin to human α7 nAChRs and [³H]cytisine to human α4β2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4β2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4β2 and α4β2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4β2 or α4β2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/β2 subunit interfaces of α4β2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.     

Natural products with calmodulin inhibitor properties

This review summarizes properties of various naturally occurring compounds with reported calmodulin (CaM)-inhibitory properties which include about 159 natural products belonging to different structural classes. Most inhibitors are alkaloid and peptide type of compounds and have been isolated from a wide variety of natural sources, including many plant species. Among the most potent natural anti-CaM substances, however, are several animal venoms and the antibiotic polymixin. The largest number of compounds described were discovered by means of enzymatic functional assays.     

杀虫药剂抗性家蝇品系乙酰胆碱酯酶基因的特征分析

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫药剂的作用靶标,这两大类杀虫药剂的广泛应用导致了昆虫对抗性的选择。靶标的修饰是某些昆虫产生抗性的分于机理,这种抗性是和AChE的变更型相关的,这些变更型的酶显示出对杀虫药剂的不被感性。利用RT-PCR和Streptavidin偶联磁珠技术从两种抗性家蝇(Musca domestica)品系D3和Kash中分别分离了AChE基因并测定了其按苷酸颅序。eDNA的可读框长2082bp．由此推导出了AChE的氨基酸顺序,通过与敏感家蝇品系Cooper的比较,发现了一些核苷酸顺序差异和4个氨基酸点突变,其中3个替代可能与杀虫药剂不敏感性有关。这一结果表明D3和Kash均属于CH₂抗性类型。     

Crystal Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHβE Suggests a Unique Mode of Antagonism

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.     

A hypothesis to account for the selective and diverse actions of neonicotinoid insecticides at their molecular targets,nicotinic acetylcholine receptors: catch and release in hydrogen bond networks

The low mammalian toxicity of neonicotinoid insecticides has been shown to be attributable, at least in part, to their selective actions on insect nicotinic acetylcholine receptors (nAChRs). There are multiple nAChRs in insects and a wealth of neonicotinoid chemicals. Studies to date have discribed a wide range of effects on nAChRs, notably partial agonist, super agonist and antagonist actions. Both the diversity of the neonicotinoid actions and their selectivity for insect over vertebrate nAChRs are the result of physicochemical and steric interactions at their molecular targets (nAChRs). In such interactions, the formation and breakage of hydrogen bond (HB) networks plays a key role. Therefore the loss or gain of even a single HB resulting from either structural changes in neonicotinoids, or the amino acid sequence of a particular nAChR subunit, could result in a drastic modification of neonicotinoid actions. In addition to the amino acid residues, the backbone carbonyl of nAChRs may also be involved in the formation of HB networks with neonicotinoids.     

Synthesis and insecticidal activity of chromanone and chromone analogues of diacylhydrazines

Diacylhydrazine derivatives have been identified as one of the most important insect growth regulators. A variety of diacylhydrazine derivatives were designed and synthesized in recent years due to their unique action mechanism, simple structure, and environmental benign character. This paper describes the molecular design, synthesis, and insecticidal activities of a series of chromanone and chromone analogues of diacylhydrazine derivatives. The preliminary bioassay showed that some of the chromanone analogues exhibited good insecticidal activity against Mythima separata at the dosage of 500 mg L-1. The present work demonstrated that replacement of the chroman ring of ANS-118, a commercial insecticide, with chromanone moiety could result in new compounds with high potent insecticidal activity.