The herbicide binding niche of photosystem II-a model

The Q_B binding niche of photosystem II is also the binding site for many different herbicides. In order to understand the mode of binding of the herbicides, a 3-dimensional model of the binding niche was constructed. The model was based upon a comparison of the known structure of the Q_B binding niche in purple bacteria with sequence and mutant data of the D-1 protein of photosystem II. Plastoquinone builds up hydrogen bonds to phenylalanine 265 backbone amide nitrogen, to serine 264 hydroxyl, and to histidine 215 delta-1 nitrogen. In addition to these hydrogen bond donors and acceptors, herbicides can build up hydrogen bonds to backbone carbonyl of alamine 263 and to serine 268 hydroxyl. This is supported by binding data of inhibitors in Chlamydomonas reinhardtii chloroplasts of wild type and of five D-1 protein mutants (Ser264 Ala, Ala251 Val, Phe255Tyr, Val2191le, Leu275Phe).     

Leaf chlorophyll fluorescence discriminates herbicide resistance in Echinochloa species

Timely detection of herbicide resistance at an early stage of crop cultivation is essential to help farmers find alternative solutions to manage herbicide resistance in their fields. In this study, maximum quantum yield of PS II [F_v/F_m = (F_m – F_o)/F_m] was measured at the 4–5 leaf stage to discriminate between herbicide‐resistant and susceptible biotypes of Echinochloa species. The differences in F_v/F_m between herbicide‐resistant and susceptible Echinochloa spp. were consistent with the whole‐plant assay based on I₅₀ (herbicide doses causing a 50% inhibition of F_v/F_m) and GR₅₀ (herbicide doses causing a 50% reduction in plant fresh weight) values and R/S ratios (herbicide resistance index), regardless of the mode of action of the tested herbicides. A PS II inhibitor caused the fastest inhibition of F_v/F_m, compared with ACCase and ALS inhibitors, after herbicide treatment. The required time for discrimination between herbicide‐resistant and susceptible Echinochloa spp. was 64 h after PS II inhibitor treatment, much shorter than those of ACCase and ALS inhibitor‐treated plants, which required 168 and 192 h respectively. The leaf chlorophyll fluorescence assay provided reliable diagnostics of herbicide resistance in Echinochloa spp. with significant time savings and convenient measurement in field conditions compared with the conventional whole‐plant assay.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Joint action of natural and synthetic photosystem II inhibitors

The inhibitory action on photosystem II of four sorgoleone analogues, isolated from Sorghum bicolor (L) Moench, and two synthetic inhibitors, diuron and bentazone, was tested by measuring oxygen evolution of thylakoid membranes. The inhibition of oxygen evolution for mixtures of inhibitors was compared with the Additive Dose Model (ADM). ADM assumes that, at a defined response level, the effect of a mixture of inhibitors can be unambiguously expressed by the potency of either of the inhibitors applied separately. The slope of the logistic dose-response curves differed between the inhibitors; sorgoleone analogues had the steepest and bentazone the shallowest slope. The difference in slopes makes the interpretation of the isoboles less general and may reflect the differences in the interaction between the natural and the synthetic inhibitors with the binding site. The results suggest that there may be some limitation to ADM, namely that compounds with the same site of action might have different response curves if their mechanism of binding is different. The joint action of inhibitors follows ADM at I₅₀ . Therefore, the inhibitors can replace each other in any mixture ratio, based on the relative potencies of the pure inhibitors, without changing each other's effect on oxygen evolution. The joint action at I₂₀ and I₈₀ sometimes diverged from ADM. © 1999 Society of Chemical Industry     

Block of electron transport by surangin B in bovine heart mitochondria

Exposure of mitochondria isolated from bovine heart to the insecticidal coumarin surangin B results in inhibition of complex II (IC₅₀ = 0.2 μM), III (IC₅₀ = 14.8 μM), and IV (IC₅₀ = 3.1 μM), but in contrast, the NADH:ubiquinone reductase (complex I) was completely insensitive to this compound at 100 μM. Kinetic analysis of surangin B’s interaction with complex II was then investigated using sub-mitochondrial particles. With succinate as the substrate, surangin B, like carboxin, acted with non-competitive kinetics and clearly contrasted in its action with malonate, a competitive inhibitor of complex II. Likewise, surangin B acted as a non-competitive inhibitor of decylubiquinone-dependent interception of electrons at complex II. Difference spectra of reduced complex III equilibrated with surangin B were found to closely parallel those of antimycin A, but were different in nature to those of the Q_o site inhibitors myxothiazol and famoxadone. Investigation of surangin B-dependent functional perturbation of complex III used the synthetic electron acceptor 2-nitrosofluorene, which intercepts electrons specifically from the Q_i site. These experiments demonstrated that like antimycin A, surangin B acts as a selective blocker of electron diversion to 2-nitrosofluorene through Q_i within complex III. We conclude that surangin B blocks electron transport at several points in bovine heart mitochondria, however, complex I is spared. The potent inhibitory action of surangin B on complex II involves binding to a site which is distinct from both the succinate binding site and the domain responsible for interacting with ubiquinone. Surangin B apparently blocks complex III by interacting with the Q_i (antimycin A-binding) pocket.     

Spirosultam LY219048: A new chemical class of insecticide acting upon the GABA receptor/chloride ionophore complex

This study assessed the toxicity and mode of action of a new experimental insecticide, LY219048 in insects and mammals. LY219048 produced rapid convulsions in mice and had LD₅₀ values of 0.7 mg kg^?1 and 4 mg kg^?1 after intracerebral and intraperitoneal injection, respectively. In initial screens against insects, LY219048 showed low activity against the German cockroach (Blatella germanica L.). Lethality from dietary exposure required one to two weeks, even at concentrations as high as 10000 mg kg^?1 (LC₅₀ = 485 mg kg^?1). In contrast, it had an LC₅₀ value of 8.3 mg kg^?1 against insecticide-susceptible Drosophila melanogaster (Meig.) when synergized with piperonyl butoxide. Significant resistance to LY219048 (> 12-fold) was detected in a cyclodiene-resistant strain of D. melanogaster possessing an altered target site resistance mechanism. This finding suggested that LY219048 blocked the 4-aminobutyric acid (GABA)-gated chloride channel in a manner similar to that of the cyclodienes. In physiological studies in larval D. melanogaster central neurons, LY219048 antagonized the reduction of firing caused by 1 mM GABA. Dose-response experiments showed that the ED₅₀ for blocking inhibition under these conditions was c. 1 μ. Studies of ³⁶CI uptake into bovine brain synaptosomes found that LY219048 was a potent antagonist. At 10 μ it completely blocked chloride flux stimulated by 50 μM GABA. LY219048 competitively displaced [³H]TBOB binding from bovine brain membranes, with an IC₅₀ of 42 nM, which was comparable to values determined for TBPS (35 nM) and picrotoxinin (267 nM). There was little or no displacement (<25%) of [³H]flunitrazepam or [³H]muscimol binding by 10 μM LY219048. Taken together, these results provide strong evidence that this new chemical class of insecticide manifests its acute toxicity by blocking the GABA-gated chloride channel.     

In vitro and in vivo analyses of the mechanism of action of SWEP

In vitro experiments with intact chloroplasts from hydroponically grown spinach (Spinacia oleracea L. var. Winter Giant) plants, have shown an I₅₀ value for SWEP (methyl N-3,4-dichlorophenyl) carbamate) of 0.1 μM in PS I and II-linked electron transport H₂0 → NADP⁺. With thylakoid membranes the I₅₀ values for PS II-linked Hill reactions H₂O → [Fe(CN)₆]³⁻ and H₂O → dichlorophenolindophenol are in the range 0.05-0.1 μM, whereas the I₅₀ shifts to 0.45 μM in short PS II-linked transport chain diphenylcarbazide → dichlorophenolindophenol. Trypsination of PS II-enriched particles produces a negligible increase of the I₅₀ value in diphenylcarbazide → dichlorophenolindophenol electron transport, a much smaller increase than occurs with diuron- or atrazine-type inhibitors. All these data show SWEP as a strong inhibitor of electron transport in the Q-B region of the PS II-reducing side. However, it appears to have a different binding site than that of urea and triazine herbicides, either on a trypsin resistant or on a non-surface cluster. As a consequence of the NADPH shortage, SWEP brings about a strong inhibition of CO₂ assimilation, with an I₅₀ of 0.04 μM, and a lower percentage of trioses-P among the intermediates of the Calvin cycle. In vivo experiments have shown a three to five times higher inhibition of PS II-linked electron transport, when SWEP was supplied through the roots than when it was applied to the leaves. We have found I₅₀ values of CO₂ assimilation by isolated chloroplasts of foliar disks of 3 and 5 μM, respectively, when the herbicide was root supplied, as opposed to 10 and 25 μM after leaf application.     

Effect of some substituted benzonitriles on photosynthetic activity of spinach and wheat in vivo and in vitro

The inhibitory effects of nine nitro and/or bromo-substituted benzonitrile compounds on the photosynthetic electron flow in isolated chloroplasts and on the in vivo CO₂ fixation of spinach (Spinacia oteracea L.) and wheat (Triticum aesticum L. cv. Bezoslaya) were investigated. Bromoxynil and 3-nitro-5-hromo-4-hydroxy-bcnzonitrile were the strongest and equally effective inhibitors of Ihe in vivo CO₂ fixation of spinach, hut in wheat the nitro-bromo-compound is ineffective and 3-nitro-benzonitrile is even more inhibitory than bromoxynil. None of the substances affected DCPIPH → methylviologen reduction. In the inhibition of the DCPIP reduction only the 3,5-disuhstituted 4-hydroxy-derivatives were effective. The fact that these compounds affect only the PS II reaction with both H₂O and DPC as electron donors suggests a site of inhibition on the reducing side of PS II, between Ihe PS II reaction centre and ihe DCPIP Site. It is suggested that in the inhibition of the DCPIP reduction only steric factors are important and the different electron configuration of the sterieally similar molecules may be involved only in the absorption and translocation processes of the compounds.     

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C_1–4 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 GABA_A receptors. © 1999 Society of Chemical Industry     

Mode of action of herbicidal ALS-inhibitors on acetolactate synthase from green plant cell cultures,yeast, and Escherichia coli

Inhibitors of branched-chain amino acid biosynthesis—by inhibiting acetolactate synthase (ALS)—represent the most active group of herbicidal compounds to date (Shaner, D.L., Recent Adv. Phytochem. 23 (1989) 227–61). A microbial screening technique has been developed to investigate known and possible new ALS-inhibitors. Escherichia coli mutant FD 1062, which expresses only valine-resistant ALS II isoenzyme as the solely branched-chain amino acid synthesizing isoenzyme, has been used extensively to optimize known and to screen for new chemical classes of ALS-inhibitors, respectively. Herbicidal compounds like sulfonylureas, triazolopyrimidines, pyrimidylsalicylates, carbamoylpyrazolines, sulfonylimino-azinyl-heteroazoles, sulfonylamide azines, and substituted sulfonyldiamides, respectively, are active on minimal medium with K_i-values which resemble the rank order of biological activity of these compounds in the greenhouse. Interestingly, herbicidal imidazolinones are not at all inhibitory on E.coli strain FD 1062 in vivo although, of course, they exert high activity on isolated bacterial ALS. Similarly, N-protected valylanilides, pyrimidyl mandelic acids, benzenesulfonyl carboxamides, and uhiquinone-O are inactive in the bacterial assay but have been shown by other methods to act as ALS inhibitors. Additionally, reversal experiments can he performed to exclude, e.g. artificial inhibitory effects of test compounds. Moreover, a thin-layer biogram application technique opens the opportunity to test mixtures of chemicals. From green plant cell cultures (Catharanthus roseus) ALS has been isolated and characterized in terms of inhibition by sulfonylureas, imidazolonones, triazolopyrmidines, salicylated, and carbamoylpyrazolines, imidazolinones, triaiolopyritnidines, salicjdates, and carbarnoylpj?razolines, respectively. All five types show biphasic slow tight binding kinetics with Stedy state I₅₀values of 0.5 nm (sulforneturon), 1.9 nm (triazolopjirimidine), 8.3 nm (salicylate), 23 nm (imazapyr), and 135 nm (carbamoylpyrazoline),respectiuely . Isolated ALS from Saccharomyces cerevisiae is equallv well blocked by herbicidal ALS inhibitors although with different I₅₀values ( triazoiopyrimidine, 21 nm, sulforneturon, 70 nm, salicylate, 21 μm, imaiapyr, 38μm, and carbamoylpyrazoline, 148 μm ). Surprisingly, biphasic kinetics could not be observed with the yeast enzjtme although slow binding hehauiour was clearly established.     

Mode of action of famoxadone

Famoxadone is a preventative and curative fungicide recently developed for plant disease control. The molecule and its oxazolidinone analogs (OADs) are potent inhibitors of mitochondrial electron transport, specifically inhibiting the function of the enzyme ubiquinol:cytochrome c oxidoreductase (cytochrome bc₁). Visible absorbance spectral studies on the purified enzyme suggested that famoxadone bound close to the low potential heme of cytochrome b. This binding mode was confirmed in competitive binding experiments by studying the displacement of a radiolabelled OAD from submitochondria. EPR studies on the binding of famoxadone to submitochondria and purified bc₁ suggested its binding mode was more like that of myxothiazol than that of stigmatellin (ligands known to bind near the low potential heme). Zoospores of Phytophthora infestans, when given low concentrations of famoxadone and other OADs, were observed to cease oxygen consumption and motility within seconds and later the cells disintegrated, releasing the cellular contents. Famoxadone was a potent inhibitor of the growth of Saccharomyces cerevisiae when grown on non-fermentable carbon sources and it was an approximately 50-fold less potent inhibitor of growth when the yeast was grown on a fermentable carbon source, glucose. Such physiological observations are consistent with the loss of mitochondrial function imposed by famoxadone and OADs. Single amino acid changes in the apocytochrome b of baker's yeast cytochrome b located near the low potential heme altered the inhibition constants for the inhibitors famoxadone, myxothiazol, azoxystrobin and kresoxim-methyl differentially, thus strongly suggesting different binding interactions of the protein with the inhibitors. © 1999 Society of Chemical Industry     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Isolation of microsomal cytochrome-p450 isozymes from Ustilago maydis and their interaction with sterol demethylation inhibitors

A procedure for the isolation of microsomes containing cytochrome-P450 isozymes from Ustilago maydis is described. Yields of P450 amount to approximately 19(±+ 6) pmol mg^?1 of microsomal protein. The wavelength of maximum absorbance of the reduced carbon monoxide difference spectrum is 448-449 nm. The azole fungicides prochloraz, etaconazole, imazalil, triadimefon and 3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazoline, which differ markedly in toxicity to U. maydis, all induce type II binding difference spectra at extremely low concentrations (10^?9-10^?8 M). The DMI concentrations which cause half saturation of type II binding difference spectra (IC₅₀) do not correlate with the fungicidal activities of the azoles. Binding of carbon monoxide to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. However, the inhibition of carbon monoxide binding also does not correlate with fungitoxicity of the DMIs. The results in this paper suggest that the spectrophotometric studies with this preparation are not useful for evaluating selective toxicity of DMIs to intact sporidia of U. maydis.     

In search of new herbicidal inhibitors of the non‐mevalonate pathway

The first five steps of the non‐mevalonate pathway have been tested in high‐throughput screening (HTS) campaigns, using enzymes of plant origin. Hit rates were in general relatively low, which could be attributed to the high polarity and charged nature of substrates and active sites of these enzymes. Still, for all the enzymes, apart from IspF (2‐methylerythritol 2,4‐cyclodiphosphate synthase), inhibitors could be identified with activities below 100 μM, and these were followed up to identify structure–activity relationships (SARs). For the enzyme IspD (2C‐methyl‐D‐erythritol 4‐phosphate cytidyltransferase), inhibitors with IC₅₀ down to 35 nM were identified that also showed herbicidal activity.     

The inhibition of chitin synthesis in spodoptera littoralis larvae by flufenoxuron,teflubenzuron and diflubenzuron

The chitin precursor [¹⁴C] N-acetylglucosamine injected into the haemolymph of Spodoptera littoralis (Boisduval) larva was incorporated into the chitin exponentially with time. When caterpillars were injected with precursor at the commencement of feeding on acylurea-treated leaf discs, flufenoxuron, teflubenzuron and diflubenzuron were found to be equally effective inhibitors of chitin synthesis, measured after 21 h. The dose response curves by feeding are not parallel, indicating that the relative potency of the compounds will vary across the dose range. When chitin precursor was injected simultaneously with topically applied diflubenzuron, flufenoxuron or teflubenzuron, all three acylureas were found to be equally effective as inhibitors of chitin synthesis when measured after five hours. The I₅₀values (50% inhibition of chitin synthesis) were not significantly different; average 600 ng, compared with LD₅₀values (50% lethal dose) of ～ 13 ng for flufenoxuron and teflubenzuron but 130 ng for diflubenzuron (topical application). Injection of precursor 24 h after topical application of insecticide gave an I₅₀value which had dropped 670- and 150-fold for flufenoxuron and teflubenzuron respectively but only 20-fold for diflubenzuron. It is postulated that the reason for the low increase in diflubenzuron effectiveness with time was due either to less diflubenzuron than flufenoxuron reaching the site of action, or more probably, a faster rate of metabolism and excretion for diflubenzuron. The lower toxicity of diflubenzuron compared with flufenoxuron and teflubenzuron may not be due to any inherent differences in biochemical effectiveness, but rather to different penetration/metabolism properties.     

Cross‐resistance of Echinochloa species to acetolactate synthase inhibitor herbicides

This study was conducted to evaluate the cross‐resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac‐sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus‐galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross‐resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac‐sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus‐galli, respectively. An in vitro ALS enzyme assay with herbicides showed that the I ₅₀‐values of the resistant accessions were approximately two‐to‐three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac‐sodium. A whole plant assay with fenitrothion showed that the GR ₅₀‐values significantly decreased in both the resistant E. oryzicola and E. crus‐galli accessions when azimsulfuron, penoxsulam and bispyribac‐sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross‐resistance for azimsulfuron, penoxsulam and bispyribac‐sodium is related to enhanced herbicide metabolism.     

Structural modifications increase the insecticidal activity of ryanodine

The toxicity of ryanodine ( 1 ) and 9,21-didehydroryanodine ( 2 ) (the principal active ingredients of the botanical insecticide ryania) to adult female house flies (Musca domestica L.) is attributable to binding to the ryanodine receptor (ryr) and thereby disrupting the Ca²⁺-release channel. These ryanoids, assayed in house flies with piperonyl butoxide (PBO) to suppress cytochrome P450-dependent detoxification, give injected KD₅₀ values of 0·07–0·11 μg g^-1, injected LD₅₀ values of 0·39–0·45 μg g^-1 and topical LD₅₀ values of 12– 50 μg g^-1. They inhibit the [³H]ryanodine binding site of house fly and rabbit muscle with IC₅₀ values of 3–10 nM . This study examines the effect of structure on potency, with 15 variants of the cyclohexane substituents, two 4,6-cyclic boron and two methylated derivatives, and four modifications of the isopropyl and ester substituents. The most effective compound examined was 10-deoxy- 2 ( 3 ) which was more potent than 2 by 2–4-fold on injection and 29-fold applied topically following PBO (LD₅₀ 0·41 μg g^-1). Additional high-potency compounds were 10-oxo- 1 and the cyclohexane variants with lactam, 21-nor-9-oxo and 21-nor-10-deoxy substituents. Other modifications usually reduced toxicity. The injected knockdown potency of the ester ryanoids was generally related to their effectiveness in competing with [³H]ryanodine at the ryr of rabbit skeletal muscle. Two non-ester ryanoids, ryanodol and 9,21-didehydroryanodol, were found to be more toxic than predicted from their potency at the ryr and may therefore act in a different manner such as at a K⁺ channel, as suggested by Usherwood and Vais. Clearly ryanoids are challenging prototypes for a potential new generation of insecticides. © 1997 SCI.     

溴氰菊酯对马铃薯腐烂茎线虫的毒力及对其运动和摄食的影响

采用药液浸渍法、沙柱法以及与荧光染料Cy3共孵育的方法,以丙溴磷、克百威和阿维菌素为对照药剂,测定了溴氰菊酯对马铃薯腐烂茎线虫Ditylenchus destructor的毒力以及对其运动扩散和摄食的影响。结果表明:溴氰菊酯对马铃薯腐烂茎线虫具有一定的杀灭活性,其LC50值为459.8mg/L,活性低于丙溴磷(159.9mg/L)、克百威(331.9mg/L)和阿维菌素(257.3mg/L);溴氰菊酯对马铃薯腐烂茎线虫运动扩散的抑制作用IC50值为3.1mg/L,其活性低于阿维菌素(0.8mg/L),但高于丙溴磷(8.3mg/L)和克百威(16.1mg/L)。当丙溴磷和克百威质量浓度分别低至0.6和40mg/L时,可刺激90%以上的线虫摄食;当丙溴磷和克百威质量浓度最低分别为360和300mg/L时,可抑制全部线虫的摄食;溴氰菊酯与阿维菌素则对线虫的摄食无刺激作用;用10mg/L的丙溴磷处理线虫2h后再分别用阿维菌素和溴氰菊酯处理,发现阿维菌素和溴氰菊酯质量浓度最低分别为20和200mg/L时可抑制全部马铃薯腐烂茎线虫的摄食。研究表明,溴氰菊酯对马铃薯腐烂茎线虫具有较高的活性,其在线虫防治领域的开发应用潜力较好。     

Synthesis and structure–activity relationship analysis of bicyclophosphorothionate blockers with selectivity for housefly γ‐aminobutyric acid receptor channels

BACKGROUND: Bicyclophosphorothionates (2,6,7‐trioxa‐1‐phosphabicyclo[2.2.2]octane‐1‐sulfides) are blockers (or non‐competitive antagonists) of γ‐aminobutyric acid (GABA) receptor channels. Twenty‐two bicyclophosphorothionates with different 3‐ and 4‐substituents were synthesised, and [³H]4′‐ethynyl‐4‐n‐propylbicycloorthobenzoate (EBOB) binding assays were performed to evaluate their affinities for housefly and rat GABA receptors. RESULTS: Introduction of an isopropyl group at the 3‐position enhanced the affinity of bicyclophosphorothionates for housefly GABA receptors and reduced the affinity towards rat GABA receptors. The 4‐isopentyl‐3‐isopropylbicyclophosphorothionate showed the highest affinity for housefly GABA receptors (IC₅₀ = 103 nM ) among the analogues tested, while the 4‐cyclohexylbicyclophosphorothionate showed the highest affinity for rat GABA receptors (IC₅₀ = 125 nM ). Among the bicyclophosphorothionates synthesised to date, the former analogue exhibited the highest selectivity for housefly GABA receptors, with an IC₅₀^rat/IC₅₀^fly ratio of approximately 97. Three‐dimensional GABA receptor models successfully explained the structure–activity relationships of the bicyclophosphorothionates. CONCLUSION: The results indicate that minor structural modifications of blockers can change their selectivity for insect versus mammalian GABA receptors. The substituent at the 3‐position of the bicyclophosphorothionates dictates selectivity for housefly versus rat GABA receptors. This information should prove useful for the design of safer insecticides and parasiticides. Copyright © 2010 Society of Chemical Industry     

Inhibition of sterol biosynthesis in cell-free extracts of Botrytis cinerea by prochloraz and prochloraz analogues

The sensitivity of cytochrome-P450-dependent sterol 14α-demethylase (P450_14DM) to prochloraz and several prochloraz analogues was studied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC₅₀ values (concentrations which inhibited radial growth of B. cinerea by 50%) of the compounds tested ranged from 3.3 × 10 ?8 to 1.7 × 10 ?5 M. The IV₅₀ values (concentrations which inhibited cell-free C₄-demethyl sterol synthesis by 50%) in cell-free assays of B. cinerea ranged from 2.6 × 10 ?9 to 4.4 × 10 ?7 M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the order of fungitoxicity, but the IC₅₀ values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition of cell-free P450_14DM activity by these compounds cannot fully account for their differences in activity towards B. cinerea. Additional mechanisms must be involved. The compounds tested were generally more potent in the B. cinerea assay than in similar assays developed for Penicillium italicum Wehmer and, in particular, Saccharomyces cerevisiae Meyen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B. cinerea is more useful to evaluate candidate fungicides as inhibitors of sterol 14α-demethyiase activity than similar assays from model organisms. The present study confirms that the affinity of prochloraz analogues for P450_14DM depends on the nature of the N-1 substituent of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4, 4-demethyl sterol biosynthesis in B. cinerea at a different site from the P450_14DM. This was confirmed by the observation that laboratory-generated triadimenol-resistant isolates of B. cinerea showed reduced sensitivity to triadimenol and prochloraz, but not to compound 6.