Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors.

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 âˆ¼20-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.

Design and biological activity of a novel fungicide, quinofumelin.

Developed by Mitsui Chemicals Agro, Inc. (Tokyo, Japan), quinofumelin is a novel fungicide with a distinct chemical structure including 3-(isoquinolin-1-yl) quinoline, demonstrating fungicidal activity against a variety of fungi, including rice blast and gray mold. We screened our compound library to identify curative compounds for rice blast and evaluated the effect of fungicide-resistant strains of gray mold. Our research demonstrated that quinofumelin has curative effects against rice blast and is not cross-resistant to existing fungicides. Accordingly, the use of quinofumelin can be considered a novel approach for disease control in agricultural production. In this report, the discovery of quinofumelin from the initial compound is described in detail.

Novel fungicide quinofumelin shows selectivity for fungal dihydroorotate dehydrogenase over the corresponding human enzyme.

The species selectivity of class 2 dihydroorotate dehydrogenase (DHODH), a target enzyme for quinofumelin, was examined. The Homo sapiens DHODH (HsDHODH) assay system was developed to compare the selectivity of quinofumelin for fungi with that for mammals. The IC50 values of quinofumelin for Pyricularia oryzae DHODH (PoDHODH) and HsDHODH were 2.8 nM and >100 µM, respectively. Quinofumelin was highly selective for fungal over human DHODH. Additionally, we constructed recombinant P. oryzae mutants where PoDHODH (PoPYR4) or HsDHODH was inserted into the PoPYR4 disruption mutant. At quinofumelin concentration of 0.01-1 ppm, the PoPYR4 insertion mutants could not grow, but the HsDHODH gene-insertion mutants thrived. This indicates that HsDHODH is a substitute for PoDHODH, and quinofumelin could not inhibit HsDHODH as in the HsDHODH enzyme assay. Comparing the amino acid sequences of human and fungal DHODHs indicates that the significant difference at the ubiquinone-binding site contributes to the species selectivity of quinofumelin.

Controlled expression of nicotinic acetylcholine receptor-encoding genes in insects uncovers distinct mechanisms of action of the neonicotinoid insecticide dinotefuran.

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 (Dα1KO) 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 Dα1KO flies, whereas the binding of dinotefuran remained unchanged. RNA sequencing analysis indicated that Dα2 expression was specifically enhanced in Dα1KO flies. These findings suggest that changes in Dα1 and Dα2 expression contribute to the differences in the insecticidal activity of dinotefuran and imidacloprid in Dα1KO flies. Overall, our findings suggest that dinotefuran acts on distinct nAChR subtypes.

The target site of the novel fungicide quinofumelin, Pyricularia oryzae class II dihydroorotate dehydrogenase.

The target site of the novel fungicide quinofumelin was investigated in the rice blast fungus Pyricularia oryzae. Quinofumelin-induced mycelial growth inhibition was reversed by orotate but not by dihydroorotate. Recovery tests suggested that the target site of quinofumelin was dihydroorotate dehydrogenase (DHODH), which catalyzes the oxidation of dihydroorotate to orotate. Quinofumelin strongly inhibited P. oryzae class 2 DHODH (DHODH II) (IC50: 2.8 nM). The inhibitory activities of mycelial growth and DHODH II were strongly positively correlated, indicating that DHODH II inhibition by quinofumelin lead to antifungal activity. A P. oryzae DHODH II gene (PoPYR4) disruption mutant (ΔPopyr4), showing the same tendency as the quinofumelin-treated wild strain in recovery tests, was constructed, and disease symptoms were not observed in rice plants infected by ΔPopyr4. Thus, DHODH II, which plays an important role in pathogenicity and mycelial growth, is found to be the target site of quinofumelin.

Application of computational methods in the analysis of pesticide target-site and resistance mechanisms.

Meta-diamide insecticides including broflanilide have a high insecticidal activity by acting on RDL GABA receptors. Both membrane potential assays and docking studies suggest that the target site of meta-diamides is different from that of conventional noncompetitive inhibitors, such as fipronil. In fact, meta-diamides are effective against cyclodiene- and fipronil-resistant pests that carry target-site mutations. Dinotefuran uniquely possesses a tetrahydrofuran ring, whereas other neonicotinoids possess aromatic rings. Moreover, dinotefuran has been reported to be effective against imidacloprid-resistant strains. A docking study predicted the weak binding of dinotefuran to cytochrome P450s which are associated with imidacloprid resistance. Metabolic assays revealed that dinotefuran was not metabolized by these cytochrome P450s. These findings suggest that the lack of metabolic activity of P450s against dinotefuran causes a low level of cross-resistance.

Synthesis and fungicidal activities of positional isomers of the N-thienylcarboxamide.

To investigate the effects of bioisosteric replacement of the phenyl group with the thienyl group, N-phenylcarboxamide and three regioisomers of N-(substituted-thienyl)carboxamide were synthesized. The inhibitory activity on the succinate dehydrogenase prepared from the gray mold Botrytis cinerea as well as the fungicidal activity against B. cinerea were evaluated. Two isomers, N-(2-substituted-3-thienyl)carboxamide and N-(4-substituted-3-thienyl)carboxamide exhibited the same level of activity as the phenyl derivative, whereas N-(3-substituted-2-thienyl)carboxamide exhibited lower activity than the phenyl derivative, suggesting that the 2-substituted-3-thienyl and 4-substituted-3-thienyl groups functioned as bioisosteres of the phenyl group in N-phenylcarboxamide, but the other did not.

Experimental and theoretical investigation of the reaction of a 3-amidothiophene derivative with various carbonyl compounds.

The reactions of a 3-amidothiophene derivative, which is a partial structure of penthiopyrad, with various carbonyl compounds were investigated. Depending on the carbonyl compound that was used as a reactant, different products (alkenes and bis-products) were obtained from the attack of the carbon at the 2-position of the 3-amidothiophene on the carbonyl compounds. Density functional theory (DFT) calculations revealed that dehydration conditions were important for the first carbonyl addition to shift the reaction toward the product, as the products are more unstable than reactants other than aldehyde. The DFT calculations also suggested that the relative stability of the alkenyl state determined whether the second bis-product formation would proceed; i.e., the relatively unstable disubstituted alkene led to bis-products, and the stable trisubstituted or conjugated alkene yielded alkenyl products.

Mechanisms underlying the selectivity of meta-diamides between insect resistance to dieldrin (RDL) and human γ-aminobutyric acid (GABA) and glycine receptors.

BACKGROUND: Meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] show high insecticide activity by acting as antagonists to the insect resistance to dieldrin (RDL) γ-aminobutyric acid (GABA) receptors. In contrast, low-level antagonist activities of meta-diamides have been demonstrated against the human GABA type A receptor (GABAA R) α1ß2γ2S, mammalian GABAA R α1ß3γ2S, and the human glycine receptor (GlyR) α1ß. Glycine residue 336 in the membrane-spanning region M3 of the Drosophila RDL GABA receptor is essential for its high sensitivity to meta-diamide 7, [3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide]. RESULTS: We examined the effects of an equivalent mutation (M288G) in spontaneously opened human GABAA R ß3 homomers using membrane potential assay. Picrotoxin and fipronil blocked spontaneously opened human GABAA Rs ß3 and ß3-M286G in a concentration-dependent manner. In contrast, meta-diamide 7 did not block spontaneously opened GABAA R ß3 homomers, although meta-diamide 7 blocked spontaneously opened GABAA R ß3-M286G homomers. In addition, inhibitory potency of meta-diamide 7 for GABA-induced membrane potential change in cells expressing GABAA R α1ß3-M286G was much higher than that in cells expressing GABAA R α1ß3. In the same way, the equivalent mutation (A288G) in GlyR α1 increased the inhibitory potency of meta-diamide 7 for GlyRs α1 and α1ß. CONCLUSION: Studies substituting an equivalent mutation (M288G) in spontaneously opening human GABAA R ß3 homomers and human GABAA Rs α1ß3 heteromers suggest that M286 in human GABAA R ß3 is important for the low sensitivity to meta-diamide 7. In this study, we summarize the mechanisms underlying the selectivity of meta-diamides between insect RDL and human GABA and glycine receptors. © 2020 Society of Chemical Industry.

Important amino acids for function of the insect Rdl GABA receptor.

BACKGROUND: Insect Rdl GABA receptor is an important insecticide target. To design a novel insecticide, studies on the structures of homologous pentameric ligand-gated ion channels provide information about important amino acids that are necessary for the function of insect Rdl GABA receptors. RESULTS: L9'A, T12'A, T13'A, T13'S, M15'S, and M15'N mutations in the Drosophila Rdl GABA receptor subunit caused the protein to spontaneously adopt the open state conformation. In contrast, the S16'A, S16'T, S17'A, and S17'H mutant homomers showed the same levels of agonist and antagonist sensitivity as the wild-type receptor. The G336M mutation in the Drosophila Rdl GABA receptor abolished the agonist activities of ivermectin and milbemectin, but the F339M mutation did not. Additionally, the F339M mutation caused spontaneous opening of the receptor. In the Drosophila Rdl model, the hydrophobic girdle plays an important role in stabilization of the closed state. Mutations which decrease hydrophobic interactions resulted in spontaneous opening, supporting the importance of the hydrophobic girdle for keeping the channel closed. Through a mutational study of transmembrane 3 (TM3) cytoplasmic domain and Rdl GABA receptor modeling, hydrophobic interactions between TM3 and TM4 and intersubunit interaction were demonstrated to be important for channel gating. Alternatively, the intrasubunit interaction between TM2 and TM3 domains were less important for channel gating in case of Drosophila Rdl GABA receptor. CONCLUSION: This study demonstrates important amino acids critical to the function of the Drosophila Rdl GABA receptor based on the mutational studies and Drosophila Rdl GABA receptor modeling approach. © 2020 Society of Chemical Industry.

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.

Further characterization of distinct high-affinity binding sites for dinotefuran in the abdominal nerve cord of the American cockroach Periplaneta americana (Blattodea).

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 [3H]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 [3H]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.

Differential metabolism of neonicotinoids by brown planthopper, Nilaparvata lugens, CYP6ER1 variants.

Imidacloprid is very effective in controlling Nilaparvata lugens Stål, which severely damages rice plants. Following heavy imidacloprid use, imidacloprid-resistant N. lugens, which showed cross-resistance to other neonicotinoids, appeared. We used the baculovirus/Sf9 expression system to express CYP6ER1 variants carrying A375del + A376G (del3) mutations, either with or without T318S mutation, which confer imidacloprid resistance in N. lugens. These CYP6ER1 variants metabolized imidacloprid but did not metabolize dinotefuran. Moreover, Drosophila expressing a CYP6ER1 variant carrying T318S + del3 mutations were resistant to imidacloprid, with a resistance ratio of 288.7, whereas the resistance ratio to dinotefuran was 3.6. These findings indicate that N. lugens has a low level of resistance to dinotefuran, and the increase of resistance is slow. We also studied the metabolism of other neonicotinoids, as well as sulfoxaflor and flupyradifurone, by CYP6ER1 variants carrying del3 mutations, either with or without the T318S mutation. Sulfoxaflor, was not metabolized by either CYP6ER1-del3 or CYP6ER1-T318Sdel3 variants. However, these variants did metabolize flupyradifurone. This study sheds light on the substrate selectivity of CYP6ER1 variants.

In vivo and in vitro evidence for the inhibition of homogentisate solanesyltransferase by cyclopyrimorate.

BACKGROUND: Cyclopyrimorate is a highly effective bleaching herbicide discovered by Mitsui Chemicals Agro, Inc. The target site was recently reported to be homogentisate solanesyltransferase (HST) in the plastoquinone (PQ) biosynthesis pathway on the basis of the number of intermediates in cyclopyrimorate-treated plants and in vitro HST assays. Here, the target site of cyclopyrimorate was further explored using both in vivo and in vitro experiments. RESULTS: The cyclopyrimorate-dependent bleaching effect on Arabidopsis thaliana was reversed by decyl PQ, suggesting that this symptom is attributable to the inhibition of PQ biosynthesis. Furthermore, homogentisate (HGA), a substrate of HST, weakly reversed the bleaching effect of cyclopyrimorate in a dose-dependent manner. We expected that the weak reversal by HGA was due to competitive inhibition by cyclopyrimorate or des-morpholinocarbonyl cyclopyrimorate (DMC), a metabolite of cyclopyrimorate in plants that exhibit higher HST-inhibitory activity as compared to cyclopyrimorate. Kinetic analysis was therefore conducted using DMC. DMC inhibited HST competitively with respect to HGA, and was a mixed non-competitive inhibitor with respect to the other substrate, farnesyl diphosphate. Moreover, neither cyclopyrimorate nor DMC inhibited 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanesyl-1,4-benzoquinone methyltransferase, which is located downstream of HST in the PQ biosynthesis pathway. CONCLUSIONS: The target site of cyclopyrimorate and DMC is HST, which is a novel target site for commercial herbicides. © 2019 Society of Chemical Industry.

Differential metabolism of neonicotinoids by Myzus persicae CYP6CY3 stably expressed in Drosophila S2 cells.

The peach-potato aphid, Myzus persicae, is a serious crop pest that has developed imidacloprid resistance, mainly through overexpression of CYP6CY3. Here, we established a metabolic assay using Drosophila S2 cells that stably expressed CYP6CY3. We found that CYP6CY3 showed metabolic activity against imidacloprid, as well as acetamiprid, clothianidin, and thiacloprid, but had no activity against dinotefuran. Our study suggested that stable gene expression in Drosophila S2 cells is useful for examining which insecticide is metabolized by P450 monooxygenases.

Differential metabolism of imidacloprid and dinotefuran by Bemisia tabaci CYP6CM1 variants.

Imidacloprid has been used to control one of most serious pests, Bemisia tabaci. However, B. tabaci has developed imidacloprid resistance mainly by over-expressing CYP6CM1. It was reported that imidacloprid-resistant B. tabaci showed no or low level of cross-resistance against dinotefuran. Here, we expressed CYP6CM1 variants using Sf9/baculovirus and/or Drosophila S2 cells and showed that CYP6CM1 variants metabolized imidacloprid but not dinotefuran. In addition, we demonstrated that imidacloprid and pymetrozine competed for a CYP6CM1 variant more efficiently than dinotefuran, using a luminescent substrate competition assay. These results suggest that lack of metabolic activity of CYP6CM1 variants against dinotefuran caused no or low level of cross-resistance.

Discovery of broflanilide, a novel insecticide.

Broflanilide (1), discovered by Mitsui Chemicals Agro, Inc., has a unique chemical structure characterized as a meta-diamide and exhibits high activity against various pests, including Lepidopteran, Coleopteran, and Thysanopteran pests. Because broflanilide has a novel mode of action, the Insecticide Resistance Action Committee (IRAC) categorized it as a member of a new group: Group 30. The meta-diamide structure was generated via drastic structural modification of a lead compound, flubendiamide (2), and the subsequent structural optimization of meta-diamides on each of its three benzene rings led to the discovery of broflanilide. In the present study, the details of the generation of meta-diamides from the lead compound and the structural optimization of meta-diamides are described.

Action mechanism of bleaching herbicide cyclopyrimorate, a novel homogentisate solanesyltransferase inhibitor.

The action mechanism of cyclopyrimorate, a novel herbicide for weed control in rice fields, was investigated. Cyclopyrimorate caused bleaching symptoms in Arabidopsis thaliana similar to those caused by existing carotenoid biosynthesis inhibitors, mesotrione and norflurazon. However, cyclopyrimorate treatment resulted in significant accumulation of homogentisate and a reduction in the level of plastoquinone. A metabolite of cyclopyrimorate, des-morpholinocarbonyl cyclopyrimorate (DMC), was detected in plants. These data suggested that cyclopyrimorate and/or DMC inhibit homogentisate solanesyltransferase (HST), a downstream enzyme of 4-hydroxyphenylpyruvate dioxygenase in the plastoquinone biosynthesis pathway. In vitro assays showed that A. thaliana HST was strongly inhibited by DMC and weakly by cyclopyrimorate, whereas other commercial bleaching herbicides did not inhibit HST. DMC derivatives showed a positive correlation between HST inhibition and in vivo bleaching activities. These results indicate that the target site of cyclopyrimorate and DMC is HST, a novel target site of commercial herbicides.

Synthesis and activities of tolprocarb derivatives against Pyricularia oryzae: relationships among the activities for polyketide synthase, melanin biosynthesis, and rice blast.

The target site of tolprocarb has been reported to be polyketide synthase (PKS). Here, we evaluated the activities for Pyricularia oryzae PKS and melanin biosynthesis as well as the control efficacy of rice blast using a series of tolprocarb derivatives. A comparison of the inhibitory activities of PKS and melanin biosynthesis revealed a linear relationship (r 2=0.90), confirming PKS as the target site of tolprocarb. A compound beyond this relationship was metabolized by P. oryzae to an inactive compound. The control efficacy of rice blast was explained using the melting point and either the inhibitory activity of PKS or melanin biosynthesis. Structure-activity analysis revealed that both end parts of tolprocarb preferred hydrophobic groups, and the chirality of the substituent in the middle part significantly influenced the activities. These relationships will provide useful information for the development of novel PKS inhibitors.

Broflanilide: A meta-diamide insecticide with a novel mode of action.

Broflanilide is a meta-diamide [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamide] that exhibits high larvicidal activity against Spodoptera litura. It has been suggested that broflanilide is metabolized to desmethyl-broflanilide and that it acts as a noncompetitive resistant-to-dieldrin (RDL) γ-aminobutyric acid (GABA) receptor antagonist. The binding site of desmethyl-broflanilide was demonstrated to be distinct from that of conventional noncompetitive antagonists such as fipronil. It has been proposed that the site of action for desmethyl-broflanilide is close to G336 in the M3 region of the Drosophila RDL GABA receptor. However, although the site of action for desmethyl-broflanilide appears to overlap with that of macrocyclic lactones, different modes of actions have been demonstrated for desmethyl-broflanilide and the macrocyclic lactones. The mechanisms underlying the high selectivity of meta-diamides are also discussed in this review. Broflanilide is expected to become a prominent insecticide because it is effective against pests with resistance to cyclodienes and fipronil.