Interaction of formamidine pesticides with insect neural octopamine receptors: Effects on radioligand binding and cyclic AMP production

The formamidines chlordimeform, demethylchlordimeform (DCDM), amitraz and BTS-2727l were tested for their respective abilities to inhibit [³H]mianserin binding in membrane preparations of cockroach brain and, in the same tissue, to stimulate octopamine-sensitive receptors coupled to adenylate cyclase. Analysis of [³H]mianserin binding indicated negative cooperativity between two binding sites with respective K_d and B_max values of 3.82 mM and 0.886 pmol (mg protein)^?1 for a high-affinity site and 218 nM and 13.56pmol (mg protein)^?1 for a low-affinity site. DCDM, BTS-27271 and amitraz inhibit [³H]mianserin binding with IC₅₀ values similar to those obtained with octopamine and the μ-adrenergic antagonist, phentolamine, whereas chlordimeform is a poor competitor for the binding sites. Similarly DCDM, BTS-27271 and amitraz elevate cyclic AMP production in brain membrane preparations in a dose-dependent manner with K_a values of 0.32 uM, 1.5 uM and 3.4 uM respectively, whereas chlordimeform was again without effect. The formamidine-mediated responses were fully additive with the evaluation of cyclic AMP obtained using the maximal concentration of dopamine but not with octopamine-mediated increases; thus the formamidine effects appear to be expressed through partial agonism of octopamine receptors that are coupled to adenylate cyclase.     

Inhibition of juvenile hormone biosynthesis and methyl farnesoate epoxidase activity by 1,5-disubstituted imidazoles in the cockroach,Diploptera punctata

Seventeen substituted imidazoles were tested as inhibitors of juvenile hormone (JH) III synthesis by cockroach corpora allata in an in-vitro radio-chemical assay. Most of these 1,5-disubstituted imidazoles were highly potent, with IC₅₀ values of less than 100nM. The compounds differed in their ability to cause an accumulation of the precursor methyl farnesoate in the glands. Four of the imidazoles were tested by topical application to previtellogenic adult females, and all caused a significant inhibition of JH synthesis and an accumulation of intraglandular methyl farnesoate for at least three days after treatment. Methyl farnesoate epoxidase activity of homogenates of corpora allata was inhibited by the compounds TH -14 and TH -27. This P450-dependent epoxidase activity was inhibited at less than 10 nM. The results show that the 1,5-disubstituted imidazoles are powerful inhibitors of the last step of juvenile synthesis in this cockroach.     

Inhibition of epoxidation of methyl farnesoate to juvenile hormone III by cockroach corpus allatum homogenates

Radiolabeled methyl farnesoate is epoxidized to juvenile hormone III by an NADPH-dependent reaction occurring in corpus allatum homogenates from the cockroach Blaberus giganteus L. Most of the enzymatically produced juvenile hormone has the 10R configuration described for previously isolated natural juvenile hormones. The unnatural 2Z geometrical isomer of methyl farnesoate is epoxidized by the above system faster than the natural 2E isomer. Several series of chemicals known to be inhibitors of mixed-function oxidases were surveyed as inhibitors of methyl farnesoate epoxidation. The anti-juvenile hormone precocene II caused negligible inhibition at 1 · 10^?4M, whereas the best inhibitor was o-bromophenoxymethyl-imidazole with an apparent I₅₀ of 4 · 10^?7M. None of the inhibitors tested were potent morphogenetic agents on Tenebrio molitor pupae, and they failed to cause precocious development of Oncopeltus fasciatus nymphs. The inhibition of in vitro juvenile hormone biosynthesis suggests the possibility of finding an anti-hormone which acts by blocking juvenile hormone biosynthesis.     

Inhibition of Tribolium gut chitin synthetase

The chitin synthetase (CS) of Tribolium castaneum gut is inhibited 50% by 0.02 μM nikkomycin and 4 μM polyoxin D, two pyrimidine nucleoside fungicides, in in vitro assays with 10-min preincubation of enzyme and inhibitor prior to substrate addition. Tribolium CS is also sensitive to inhibition by the pyrimidine nucleotides uridine and cytidine di- and triphosphates. Captan, a known inhibitor of insect chitin synthesis, and the related fungicides captafol and dichlofluanid are highly potent inhibitors of Tribolium CS. Moderately active CS inhibitors are the acaricide oxythioquinox and the herbicide barban. One phenylcarbamate insect growth reatardant, H-24108, is weakly active in inhibiting Tribolium gut CS, as are three of its analogs but not 26 others. Many triazines are not inhibitory including several herbicides and an azido derivative, CGA 19255, which is active in blocking insect growth and chitin synthesis. Although the benzoylphenyl urea insecticides diflubenzuron and SIR 8514 are potent in vivo inhibitors of the polymerization step in insect chitin synthesis, they do not affect T. castaneum gut CS activity in vitro and greatly stimulate Tribolium brevicornis gut CS activity in vivo. These studies and preliminary findings on an integumental enzyme indicate that CS of these tissues is not sensitive to the direct action of benzoylphenyl ureas. This leads to speculation that the benzoylphenyl ureas act either as CS inhibitors via active metabolites formed in the integument or as blocking agents by direct binding to non-CS sites important in chitin polymerization and fibrillogenesis.     

The carboxyterminal processing protease of D1 protein: Herbicidal activity of novel inhibitors of the recombinant and native spinach enzymes

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for the discovery of a general broad-spectrum herbicide. Directed and random screening of compounds against recombinant spinach CtpA (rCtpA) has led to the discovery of five different chemical classes of inhibitors. Lead compounds from each inhibitor class were investigated for their in vitro effects on the activities of both recombinant and native spinach CtpA. All of the lead compounds have K_i values of less than 50 μM when tested against rCtpA, and all except one showed competitive inhibition. Results from partially purified native CtpA from spinach were similar to those from the recombinant form of the enzyme, thus validating the use of rCtpA in the inhibitor screen. Compounds from three of the classes of CtpA inhibitors show in vivo herbicidal activity against Arabidopsis thaliana when applied either by addition to growth media or by spraying the leaves. Transgenic Arabidopsis plants which over-express CtpA showed greater resistance to the compounds than wild-type plants providing evidence that these inhibitors are directly acting against CtpA.     

Aryl N-hydroxy- and N-methoxy-N-methylcarbamates as potent reversible inhibitors of acetylcholinesterase

Several aryl N-hydroxy- and N-methoxy-N-methylcarbamates were examined as inhibitors of bovine erythrocyte acetylcholinesterase (AChE). These carbamate derivatives were generally strong inhibitors of AChE, but, unlike the typical N-methyl- and N,N-dimethylcarbamates which are carbamylating agents, they proved to be reversible, competitive inhibitors of the enzyme. The values for the dissociation constant (K_a) for the enzyme-inhibitor complex to enzyme and inhibitor were in the range of 2 × 10^?5?1 × 10^?7M.     

Effects of diazepam and chlordimeform analogs on the German and the American cockroaches

Twenty-one diazepam- and chlordimeform (CDM)-related compounds were synthesized by mimicking some parts of the 1,4-benzodiazepine tranquilizing drugs, and were tested for their insecticidal activity against the German cockroach. Some of these compounds showed knockdown effects and some were insecticidal. Against the German cockroach the most toxic CDM analog was N-propargyl CDM (compound 6), and that with a potent knockdown potency was compound 13 which has a structural resemblance to diazepam. Ligand-receptor binding assay was carried out, using [³H]diazepam as a ligand to examine the relation between CDM-related compounds and the 1,4-benzodiazepines. The [³H]diazepam binding to a specific site in the American cockroach brain was inhibited by the insecticidal compounds. Among these compounds a correlation exists between their inhibitory potency on specific [³H]diazepam binding and their insecticidal activity, suggesting a possible significance of such an interaction with the diazepam binding site for the toxicity of these compounds against cockroaches.     

Inhibition of termite cellulases by carbohydrate-based cellulase inhibitors: Evidence from in vitro biochemistry and in vivo feeding studies

Efficacy of three prototype termite cellulase inhibitors, cellobioimidazole (CBI), fluoromethylcellobiose (FMCB) and fluoromethylglucose (FMG) was investigated using biochemical and feeding assays. Optimal conditions for measuring endoglucanase, exoglucanase and β-glucosidase activities were first determined. The three inhibitors were then tested under optimal conditions against enzyme fractions that represented endogenous (foregut/salivary gland/midgut) and symbiotic (hindgut) cellulases. In vitro, CBI and FMCB both inhibited exoglucanase and β-glucosidase activity (I₅₀s in nM and mM range, respectively). Feeding assays showed significant impacts on both survivorship and feeding stimulation by FMCB and CBI. Enzymatic measurements on feeding assay survivors showed impacts on all three cellulase activities by CBI and lesser impacts by FMG and FMCB. Validative bioassays with the sugars glucose, maltose and cellobiose showed no feeding stimulation or mortality as occurred in feeding inhibitor bioassays. These results indicate efficacy for two cellobiose-based inhibitors, FMCB and CBI, suggesting potential for these inhibitors as novel termite control agents.     

Selective inhibition of JH esterases from cockroach hemolymph

Juvenile hormone III was tritium labeled on the methyl ester and utilized with other substrates in an investigation of inhibition and substrate specificity of hemolymph esterases from the cockroach, Blaberus giganteus. The structure of labeled juvenile hormone III was supported both chemically and biochemically. Forty-two potential inhibitors were examined, and the best inhibitors included phosphoramidothiolates and S-phenylphosphates. One of these inhibitors was found useful in hormone biosynthesis studies dealing with the enzymatic conversion of methyl farnesoate to juvenile hormone in corpora allata homogenates. Several commonly used inhibitors of carboxyesterases caused only weak inhibition of JH esterases. Gel filtration elution patterns, inhibitor relationships, and specific activities of the hemolymph esterases indicate that juvenile hormones I and III are degraded by similar if not identical enzymes. In some cases, α-naphthyl acetate and juvenile hormone esterase activity could be differentially inhibited. Hemolymph esterases were not capable of degrading ethyl or isopropyl conjugated esters of two juvenoids or three model substrates.     

Firefly luciferase inhibition by isopropyl-3-chlorocarbanilate and isopropyl-3-chloro-hydroxycarbanilate analogues

The firefly luciferase ATP assay was inhibited by the herbicide, isopropyl-3-chlorocarbanilate (I), and by two of its hydroxylated metabolites, isopropyl-5-chloro-2-hydroxycarbanilate (II) and isopropyl-3-chloro-4-hydroxycarbanilate (III). The β-O-glucosides of II and III reversed the inhibition of luciferase. Compounds I and II were linear noncompetitive inhibitors in respect to ATP (K_i ? 20 μM, each) and were linear competive inhibitors in respect to d-luciferin (K_i ? 6 μM, each). Compound III was a linear competitive inhibitor in respect to both ATP and d-luciferin (K_i ? 1 and 6 μM, respectively). The inhibition caused by III appeared to remain competitive for both substrates when AMP was added to the system, but the inhibition exhibited by III with respect to ATP and d-luciferin was more effective (K_i ? 0.5 μM, each). The effects of compounds I, II, and III upon the firefly luciferase ATP assay are discussed, and a relationship between the firefly system and plant susceptibility to compound I is proposed.     

Various mechanisms responsible for permethrin metabolic resistance in seven field-collected strains of the German cockroach from Iran, Blattella germanica (L.) (Dictyoptera: Blattellidae)

Insecticide resistance in the German cockroach can be mediated by a number of mechanisms, the most common being enhanced enzymatic metabolism. Seven field-collected strains of German cockroach, Blattella germanica (L.) with various levels of resistance to pyrethroids, five out of which were also cross-resistant to DDT were used in this study. The investigation of possible mechanisms responsible for permethrin resistance was carried out using the synergists PBO, DEF and DMC and biochemical assays, including general esterases, glutathione S-transeferases and monooxygenases assays, using an automated microtitre plate reader. PBO and DEF, the inhibitors of cytochrome p450 monooxygenases and general esterases, respectively, affected permethrin resistance to varying degrees depending on the strain. DDT resistance in five strains were not completely eliminated by the synergist DMC, an inhibitor of glutathione S-transferase enzymes, suggesting that a further non-metabolic resistance mechanism such as kdr-type may be present. This suggestion was further supported by GST assay data, where a little elevation in GST activity was detected in only two strains. The synergist data supported by biochemical assays implicated that cytochrome p450 monooxygenases or hydrolases are involved in permethrin resistance in some strains. However, these results implicated both enhanced oxidative and hydrolytic metabolism of permethrin as resistance mechanism in the other strains. The results of synergist and biochemical studies implicated that all the field-collected permethrin resistant strains have developed diverse mechanisms of resistance, although these strains have been collected from the same geographic area. The change in resistance ratios of some strains by using PBO or DEF is discussed. It is of interest to note that because resistance to permethrin was not completely eliminated by DEF and PBO, it is likely that one or more additional mechanisms are involved in permethrin resistance in every strain studied.     

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.     

Catabolism of tryptamine by cockroach head enzyme preparation

An enzyme preparation from the homogenate of American cockroach heads [Periplaneta americana (L.)] converted tryptamine hydrochloride to a toluene-extractable material. Optimum conditions for the reaction were obtained with 0.3 M sodium phosphate buffer, pH 6.6, at 30°C. The enzyme was very unstable. Its activity was scarcely inhibited by such typical monoamine oxidase inhibitors as iproniazid phosphate and tranylcypromine hydrochloride. Enzyme activity was not affected by the addition of NADPH₂, but was accelerated by acetyl coenzyme A or coenzyme A. A main product of the enzymic reaction was isolated and identified as N-acetyltryptamine by chromatography and spectroscopic analyses, which suggests that the enzyme is a kind of N-acetyltransferase.     

Purification, characterization and inhibition studies of α-amylase of Rhyzopertha dominica

Rhyzopertha dominica causes extensive damage to stored wheat grains. α-Amylase, the major insect digestive enzyme, can be an attractive candidate to control the insect damage by inhibiting the enzyme through α-amylase inhibitors. R. dominica α-amylase (RDA) was purified to homogeneity by differential ammonium sulphate fractionation, Sephadex G-25 and Sephadex G-100 column chromatography. The homogenous α-amylase has a molecular weight of 52 kDa. The pH optima was 7.0 and temperature optima was 40 °C. Activation energy of RDA was 3.9 Kcal mol⁻¹. The enzyme showed high activity with starch, amylose and amylopectin whereas dextrins were the poor substrates. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The enzyme showed Km of 0.98 mg ml⁻¹ for starch as a substrate. Citric acid, oxalic acid, salicylic acid, HgCl₂, tannic acid and α-amylase inhibitors from wheat were inhibitors whereas; Ca²⁺ and Mg²⁺ were the activators of RDA. Ki values calculated from Dixon graphs with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitors were 6.9, 2.6-8.2, 3.2 mM and 0.013-0.018 μM, respectively. The Lineweaver-Burk plots with different inhibitors showed mixed type inhibition. Wheat α-amylase inhibitor showed mainly competitive inhibition with some non-competitive behaviour and other inhibitors showed mainly non-competitive inhibition with some un-competitive behaviour. Feeding trials with salicylic acid, citric acid, oxalic acid and wheat α-amylase inhibitor showed significant effect of salicylic acid and oxalic acid along with wheat α-amylase inhibitor in controlling the multiplication of R. dominica.     

Inhibition of the enzymatic hydration of the epoxide HEOM in insects

Enzymatic epoxide hydration, a significant mechanism in the regulation of insect development and in the detoxication of certain cyclodiene insecticides, has been investigated in vitro in the blowfly, Calliphora erythrocephala, the southern armyworm, Prodenia eridania, and the Madagascar cockroach, Gromphadorhina portentosa. Characterization of the hydrases involved in cyclodiene epoxide hydration has been achieved using as substrate a cyclodiene insecticide (HEOM) susceptible to enzymatic epoxide ring cleavage. The enzymes, which are microsomal but different from the oxidases, are inhibited in varying degrees by microsomal oxidase inhibitors as well as by certain epoxides, esterase inhibitors and compounds with reported juvenilizing ability. Group-bulk and electronegative effects are important requirements for HEOM-hydrase inhibition, the best inhibitor of the system being 1,1,1-trichloro-2,3-epoxypropane. Differences between the structural requirements for HEOM-hydrase inhibition and those for inhibition of the epoxide hydrase responsible for the degradation of juvenile hormone are discussed.     

Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase

A series of 27 substituted thio-1,1,1-trifluoropropanones was synthesized by reacting the corresponding thiol with 1,1,1-trifluoro-3-bromopropanone. The resulting sulfides were screened as inhibitors of hemolymph juvenile hormone esterase and α-naphthyl acetate esterase activity of the cabbage looper, Trichoplusia ni, electric eel acetylcholinesterase, bovine trypsin, and bovine α-chymotrypsin. The presence of the sulfide bond increased the inhibitory potency on all of the enzymes tested when compared with compounds lacking the sulfide. In general, the compounds proved to be poor inhibitors of chymotrypsin and moderate inhibitors of trypsin. By varying the substituent on the sulfide, good inhibitory activity was obtained on α-naphthyl acetate esterase, acetylcholinesterase, while some of the compounds proved to be extremely powerful inhibitors of juvenile hormone esterase. The most powerful inhibitor tested was 3-octylthio-1,1,1-trifluoro-2-propanone, with an I₅₀ of 2.3 × 10^?9M on JH esterase. This compound showed a molar refractivity similar to that of the JH II backbone, was not toxic to T. ni, and was moderately toxic to mice, with a 48-hr LD₅₀ of >750 mg/kg. It effectively delayed pupation when applied to prewandering larvae of T. ni, as expected for a JH esterase inhibitor. Thus, some members of this series are promising for evaluating the role of JH esterase in insect development. The series also indicates that, by varying the substituent on the sulfide moiety, potent “transition-state” inhibitors can be developed for a wide variety of esterases and proteases.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Comparative insecticidal activities and metabolic rates of lindane and its hexadeuterio-analog

Hexadeuterio-lindane(γ-BHC-d₆) was several times as toxic as lindane against the mosquito (Culex pipiens pallens), house fly (Musca domestica), German cockroach (Blattella germanica) and American cockroach (Periplaneta americana). The neuroexcitatory activity of these two compounds did not differ. Lindane was considerably synergized by piperonyl butoxide, but lindane-d₆ was not. A large isotope effect was observed in the in vivo breakdown of lindane-d₆. Thus, the intrinsic toxicities of both compounds are equivalent. The difference in insecticidal activity seems to be due to the different rates of detoxifying biodegradation caused by the kinetic deuterium isotope effect.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

Picrotoxinin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides

The nature of the picrotoxinin receptor was studied using the central nervous system (CNS) of the American cockroach. It first was confirmed by using an electrophysiological technique that the abdominal nerve cord of the American cockroach was sensitive to picrotoxinin. By using a $\text{[}{}^3\text{H]α-dihydropicrotoxinin}$ binding test it was determined that the picrotoxinin receptor in CNS of this insect had a higher affinity toward picrotoxinin and heptachlor epoxide than the corresponding receptor in the rat brain. Also, the cockroach brain preparation had a higher percentage of specific binding in the total binding, making this material suitable for receptor studies. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 to 1.2 M sucrose at 100,000g contained the highest level of specific binding site. The receptor showed a sensitivity to all insecticidal cyclodienes tested, namely photodieldrin, oxychlordane, endrin, heptachlor epoxide, γ-chlordane, dieldrin, aldrin, heptachlor, and isodrin (expressed in the order of potency). Among four BHC isomers, the γ-isomer showed the highest potency to bind with this receptor.