Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: in vivo and modelling/docking experiments

BACKGROUND: Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programmes. Diacylhydrazine (DAH)‐based ecdysone agonists, also known as moulting‐accelerating compounds (MACs), are considered to be a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). RESULTS: In this project, in vivo toxicity assays demonstrated that the DAH‐based RH‐5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand‐binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced, and a homology protein model was constructed that confirmed a cavity structure with 12 α‐helices, harbouring the natural insect moulting hormone 20‐hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH‐based insecticides owing to a restricted extent of the ligand‐binding cavity of the EcR of O. laevigatus. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modelling/docking experiments are indications that these pesticides do not bind with the LBD‐EcR of O. laevigatus and support the supposition that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programmes. Copyright © 2012 Society of Chemical Industry     

Comparison of the activity of non‐steroidal ecdysone agonists between dipteran and lepidopteran insects,using cell‐based EcR reporter assays

BACKGROUND: Diacylhydrazine (DAH) analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects, as does the natural insect moulting hormone 20‐hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects, raising the question as to whether non‐lepidopteran‐specific analogues can be isolated. However, for the discovery of ecdysone agonists that target other important insect groups such as Diptera, efficient screening systems that are based on the activation of the EcR are needed. RESULTS: In this study, a dipteran‐specific reporter‐based screening system with transfected S2 cells of Drosophila melanogaster Meig. was developed in order to discover and evaluate compounds that have ecdysone agonistic or antagonistic activity. A library of non‐steroidal ecdysone agonists containing different mother structures with DAH and other related analogues such as acylaminoketone (AAK) and tetrahydroquinoline (THQ) was tested. None of the compounds tested was as active as 20E. This is in contrast to the very high activity of several DAH and AAK congeners in lepidopteran cells (Bombyx mori L.‐derived Bm5 cells). The latter agrees with a successful docking of a DAH, tebufenozide, in the binding pocket of the lepidopteran EcR (B. mori), while this was not the case with the dipteran EcR (D. melanogaster). Of note was the identification of two THQ compounds with activity in S2 but not in Bm5 cells. Although marked differences in activity exist with respect to the activation of EcR between dipterans and lepidopterans, there exists a positive correlation (R = 0.724) between the pLC₅₀ values in S2 and Bm5 cells. In addition, it was found through protein modelling that a second lobe was present in the ligand‐binding pocket of lepidopteran BmEcR but was lacking in the dipteran DmEcR protein, suggesting that this difference in structure of the binding pocket is a major factor for preferential activation of the lepidopteran over the dipteran receptors by DAH ligands. CONCLUSIONS: The present study confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show this specificity, indicating that dipteran‐specific ecdysone‐agonist‐based insecticides based on the THQ mother structure can be developed. The differences in activity of ecdysone agonists in dipteran and lepidopteran ecdysone‐reporter‐based screening systems are discussed. Copyright © 2010 Society of Chemical Industry     

Ecdysteroid receptor docking suggests that dibenzoylhydrazine-based insecticides are devoid of any deleterious effect on the parasitic wasp Psyttalia concolor (Hym. Braconidae)

BACKGROUND: The moulting accelerating compounds (MACs) or ecdysteroid agonists represent a selective group of insecticides acting upon binding to the ecdysteroid receptor (EcR) and leading to lethal premature moulting in larval stages and aborted reproduction in adults. Psyttalia concolor Szèpl. is a useful parasitic wasp attacking important tephritid pests such as the medfly and olive fruit fly. RESULTS: Contact and oral exposure in the laboratory of female parasitic wasps to the dibenzoylhydrazine‐based methoxyfenozide, tebufenozide and RH‐5849 did not provoke negative effects. No mortality and no reduction in beneficial capacity were observed. The ligand‐binding domain (LBD) of the EcR of P. concolor was sequenced, and a homology protein model was constructed which confirmed a cavity structure with 12 α‐helices, harbouring the natural insect moulting hormone 20‐hydroxyecdysone. However, a steric clash occurred for the MAC insecticides owing to a restricted extent of the ligand‐binding cavity of the PcLBD‐EcR, while they did dock well in that of susceptible insects. CONCLUSIONS: The insect toxicity assays demonstrated that MACs are selective for P. concolor. The modelling/docking experiments are indications that these insecticides do not bind with the LBD‐EcR of P. concolor and support the theory that they show no biological effects in the parasitic wasp. These data may help in explaining the compatible use of MACs together with parasitic wasps in IPM programmes. Copyright © 2012 Society of Chemical Industry     

Mode of action of methoprene in affecting female reproduction in the African malaria mosquito,Anopheles gambiae

BACKGROUND: One of the most studied actions of juvenile hormone (JH) is its ability to modulate ecdysteroid signaling during insect development and metamorphosis. Previous studies in mosquitoes showed that 20‐hydroxyecdysone (20E) regulates vitellogenin synthesis. However, the action of JH and its mimics, e.g. methoprene, on female reproduction of mosquitoes remains unknown. RESULTS: Here, a major malaria vector, Anopheles gambiae Giles, was used as a model insect to study the action of methoprene on female reproduction. Ecdysteroid titers and expression profiles of ecdysone‐regulated genes were determined before and after a blood meal. An ecdysteroid peak was detected at 12 h post blood meal (PBM). The maximum expression of ecdysone‐regulated genes, such as ecdysone receptor (EcR), hormone receptor 3 (HR3) and vitellogenin (Vg) gene, coincided with the ecdysteroid peak. Interestingly, topical application of methoprene at 6 h PBM delayed ovarian development and egg maturation by suppressing the expression of ecdysone‐regulated genes in female mosquitoes. CONCLUSION: The data suggest that ecdysteroid titers are correlated with Vg synthesis, and methoprene affects vitellogenesis by modulating ecdysteroid action in A. gambiae. Copyright © 2010 Society of Chemical Industry     

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

BACKGROUND: Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR) might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine‐based compounds methoxyfenozide, tebufenozide and RH‐5849 showed different results. Methoxyfenozide and tebufenozide did not provoke any negative effects on the adults, but RH‐5849 killed 98‐100% of the treated insects 15 days after treatment. The ligand‐binding domain (LBD) of the EcR of B. oleae (BoEcR‐LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand‐binding cavity of the BoEcR‐LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH‐5849. CONCLUSION: IGR molecules similar to the RH‐5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest. Copyright © 2012 Society of Chemical Industry     

Biological activity of natural phytoecdysteroids from Ajuga iva against the sweetpotato whitefly Bemisia tabaci and the persea mite Oligonychus perseae

BACKGROUND: Ecdysteroids are steroid hormones that control moulting and govern several changes during metamorphoses in arthropods. The discovery of the same molecules (phytoecdysteroids) in several plant species displayed a wide array of rather beneficial agricultural impact. Many representatives of the genus Ajuga plants contain phytoecdysteroids with a 5β‐7‐ene‐6‐one system exhibiting physiological activities in insects. RESULTS: By means of chromatographic (silica gel column, TLC) and LC‐MS, two major ecdysteroids (20‐hydroxyecdysone and cyasterone) have been isolated and identified from Israeli carpet bugle Ajuga iva (L.) Schreber (Lamiales: Lamiaceae) plants. Ajuga iva extract fractionated on the silica gel column yielded two fractions that showed high activity against the sweetpotato whitefly Bemisis tabaci and the persea mite Oligonychus perseae. A dose of 5 mg AI L^?1 of the purely identified A. iva ecdysterone significantly reduced fecundity, fertility and survival of these pests, while commercial 20‐hydroxyecdysone at the same dose had lesser effects. CONCLUSION: The results demonstrate considerable efficacy of natural phytoecdysteroids against major agricultural pests, and suggests that these materials should be considered for potential development of friendly control agents. Copyright © 2011 Society of Chemical Industry     

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     

Cytochrome P-450 Inducers and Inhibitors Interfering with Ecdysone 20-Monooxygenases and Their Activities during Postembryonic Development of Neobellieria bullata Parker

The cytochrome P-450-dependent microsomal and mitochondrial ecdysone 20-monooxygenase systems convert ecdysone into 20-hydroxyecdysone. The microsomal fraction of fat bodies of zero h wandering stage fleshfly larvae (Neobellieria bullata; Diptera: Sarcophagidae) has a high ecdysone 20- monooxygenase activity. The effects of cytochrome P-450 inhibitors were investigated in vitro on microsomal ecdysone 20-monooxygenase. Metyrapone, fenarimol and certain imidazole derivatives (KK-42, KK-110, KK-135 and PIM) are strong inhibitors. The IC₅₀ value of KK-110, which is the strongest inhibitor, is 2 × 10^?7 M. A triazolyl and two cyclopropylamine derivatives have low activity. The activities of different NADPH-cytochrome c (P-450) reductase inhibitors were also assessed; diquat dibromide is a moderate inhibitor of microsomal ecdysone 20-monooxygenase, while paraquat dichloride has no activity. In-vivo experiments with cytochrome P-450 inducers and inhibitors gave the following results: (a) fenarimol, FI-121, precocene-2 caused “permanent” first-instar larvae; (b) barbital, phenobarbital and their sodium salts caused significant delay in larval development; (c) PIM, PTM, metyrapone, KK-42, KK-135, J-2710, RH 5849 and colchicine caused moulting disturbances; (d) J-2710, PIM, PTM, KK-42, KK-135, RH 5849 and colchicine caused lethal spiracle and mandible malformation; (e) KK-110, fenarimol, barbital and phenobarbital caused precocious pupariation.     

Nonsteroidal ecdysone receptor agonists use a water channel for binding to the ecdysone receptor complex EcR/USP

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.     

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays

BACKGROUND: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species‐specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS: When collections of non‐steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100‐fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION: The present study demonstrated that species‐specific ecdysone‐agonist‐based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification. Copyright © 2010 Society of Chemical Industry     

Receptor-binding affinity and larvicidal activity of tetrahydroquinoline-type ecdysone agonists against Aedes albopictus

Tetrahydroquinolines (THQs), a class of nonsteroidal ecdysone agonists, are good candidates for novel mosquito control agents because they specifically bind to mosquito ecdysone receptors (EcRs). We have recently performed quantitative structure–activity relationship (QSAR) analyses of THQs to elucidate the physicochemical properties important for the ligand–receptor interaction. Based on previous QSAR results, here, we newly synthesized 15 THQ analogs with a heteroaryl group at the acyl moiety and evaluated their binding affinity against Aedes albopictus EcRs. We also measured the larvicidal activity of the combined set of previously and newly synthesized compounds against A. albopictus to examine the contribution of receptor-binding to larvicidal activity. Multiple regression analyses showed that the binding affinity and the molecular hydrophobicity of THQs are the key determinants of their larvicidal activity.     

The differential binding and biological efficacy of auxin herbicides

Background

Auxin herbicides have been used for selective weed control for 75 years and they continue to be amongst the most widely used weed control agents globally. The auxin herbicides fall into five chemical classes, with two herbicides not classified, and in all cases it is anticipated that recognition in the plant starts with binding to the Transport Inhibitor Response 1 (TIR1) family of auxin receptors. There is evidence that some classes of auxins act selectively with certain clades of receptors, although a comprehensive structure–activity relationship has not been available.

Results

Using purified receptor proteins to measure binding efficacy we have conducted quantitative structure activity relationship (qSAR) assays using representative members of the three receptor clades in Arabidopsis, TIR1, AFB2 and AFB5. Complementary qSAR data for biological efficacy at the whole-plant level using root growth inhibition and foliar phytotoxicity assays have also been analyzed for each family of auxin herbicides, including for the afb5-1 receptor mutant line.

Conclusions

Comparisons of all these assays highlight differences in receptor selectivity and some systematic differences between results for binding in vitro and activity in vivo. The results could provide insights into weed spectrum differences between the different classes of auxin herbicides, as well as the potential resistance and cross-resistance implications for this herbicide class. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Quantitative structure–activity studies of insect growth regulators xiv. Three-dimensional quantitative structure–activity relationship of ecdysone agonists including dibenzoylhydrazine analogs

N-tert-Butyl-N,N′-dibenzoylhydrazines such as tebufenozide (RH-5992) mimic the action of a molting hormone, 20-hydroxyecdysone, and cause premature molting of larvae leading to their death. Previously, it was shown that one of the benzoyl moieties in dibenzoylhydrazines plays a role similar to that of the aliphatic side chain at the C-17 position of ecdysones. In the present study, N-benzoyl-N′-benzylhydrazine, N,N′-dibenzylhydrazine, and N-alkanoyl-N′-benzoylhydrazine analogs have been synthesized to compare the effects of two carbonyl groups as well as two benzene rings of dibenzoylhydrazine. The quantitative structure–activity relationship of ecdysone agonists including dibenzoylhydrazine analogs was analyzed three-dimensionally using the CoMFA (comparative molecular field analysis) procedure. The CoMFA results suggested that the two carbonyl oxygen atoms of the diacylhydrazine skeleton probably correspond to the oxygens of the 20- and 22-OH groups of ecdysones, and that the benzoyl moiety located closer to the tert-butyl group is important for retaining high activity. © 1998 SCI     

The mode of action of RH‐1965: a new phenylpyrimidinone bleaching herbicide

RH‐1965 is a new bleaching herbicide which causes newly developing leaf tissue to emerge devoid of photosynthetic pigments. Mode‐of‐action studies revealed that RH‐1965 inhibited the accumulation of both total chlorophyll and β‐carotene. Concomitantly, it induced the accumulation of the β‐carotene precursors, phytoene, phytofluene and, in particular, ξ‐carotene. Inhibition of chlorophyll accumulation by RH‐1965 is attributed to the photo‐oxidative destruction of chlorophyll in the absence of β‐carotene because RH‐1965 blocked chlorophyll accumulation to a greater extent under high light (50–330 µE m⁻² s⁻¹) than under low light (0.8 µE m⁻² s⁻¹) conditions. Radish (Raphanus sativus L) and barnyardgrass (Echinochloa crus‐galli (L) Beauv) were very senstive to RH‐1965. Under high light (330 µE m⁻² s⁻¹), the I₅₀ values for inhibition of chlorophyll accumulation were 0.10 and 0.15 µM , respectively. Wheat (Triticum aestivus L), on the other hand, was much less sensitive to RH‐1965 (I₅₀ = 1.4 µM ). It is concluded that the mode of action of RH‐1965 involves the inhibition of ξ‐carotene desaturation. © 2000 Society of Chemical Industry     

Predation and parasitism on eggs of two pod‐sucking bugs,Nezara viridula and Piezodorus hybneri in soybean

Abstract

Cohorts of newly laid egg masses of two pod‐sucking bugs, Nezara viridula and Piezodorus hybneri (Hemiptera: Pentatomidae), were exposed to naturally occurring predators and parasitoids on soybean in unsprayed fields at four sites in Northern Sumatra. The fate of the young stages was recorded until the late first instar, before the nymphs start to disperse and feed. Partial life tables showed that mortality of N. viridula until the late first instar was 50–87%, of which 18–65% occurred during the egg stage and was mainly caused by predation; 2–26% of the eggs died of parasitism. P. hybneri which was present at only two sites, showed a 73–89% mortality until the late first instar, of which 69–85% was during the egg stage and, again, mainly caused by predation; 15–24% of the eggs died of parasitism. To evaluate the role of individual egg predator species in the field, we conducted hourly observations of exposed egg masses of the two pests. Dolichoderus sp., Solenopsis geminata (Hymenoptera: Formicidae), Paederus sp. (Coleoptera: Staphylinidae) and several gryllids (Orthoptera) were most commonly encountered feeding on the eggs. An index of ‘predation pressure”; was calculated from the densities of predators in the field and from an estimate of their attack rate based on field observations of predator feeding. Two sites with low predator pressures had low predation rates, while the two sites with high predation pressures had considerably higher predation rates. The implications of the findings for IPM training in soybean are discussed.     

A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures,followed by modeling of ecdysteroid-EcR interactions and normal mode analysis

Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC₅₀ = 3.3 μM) and Bm5 cells (EC₅₀ = 5.3 μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC₅₀ = 0.71 μM and 0.00089 μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC₅₀ of 0.039 μM; in Bm5 cells this effect only became visible at much higher concentrations (IC₅₀ = 18 μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.     

Muscarinic receptor in house fly brain and its interaction with chlorobenzilate

Membranes from house fly heads were tested for the presence of mucarinic acetylcholine receptors using as a probe [³H]quinuclidinyl benzilate ([³H]QNB). Based on the presence of saturable and reversible high-affinity binding of [³H]QNB, which is inhibited by muscarinic drugs, it is suggested that these sites may be muscarinic receptors. However, these putative muscarinic receptors differ in several characteristics from the ones in mammalian brain. They have lower affinities for muscarinic drugs and lower stereoselectivity, a relatively higher affinity for the nicotinic antagonist d-tubocurarine, a lower Hill coefficient for binding of muscarinic antagonists, and a lower concentration relative to α-bungarotoxin binding sites in the same membranes. Also, unlike mammalian muscarinic receptors, they are sensitive to treatments with N-ethylmaleimide and 5,5′-dithiobis(2-nitrobenzoic acid). The effect of reduction of disulfide bonds by dithiothreitol or mercaptoethanol suggests that only the insect receptor has one or more disulfide bonds which are important to binding. On the other hand, the putative muscarinic receptors of both insect and mammalian brains have important SH group(s), whose alkylation by p-chloromercuribenzoate inhibits binding. Also, chlorobenzilate is equally effective in inhibiting [³H]QNB binding to muscarinic putative receptors of house fly and bovine brains.