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.     

DDT and pyrethroids: Receptor binding and mode of action in the house fly

The mode of action of DDT and pyrethroids was investigated in the house fly, Musca domestica L, using drug:receptor binding techniques. Both in vivo and in vitro binding studies demonstrated the existence of membrane receptors which bind specifically to [¹⁴C]DDT and [¹⁴C]cis-permethrin. The receptors show properties to be expected of a critical target site of these insecticides. These include negative temperature correlation with binding, relatively nonsensitivity to DDE, and sensitivity to Ca²⁺. The receptor sites are readily saturated at 45–90 nM [¹⁴C]DDT and have an apparent disassociation constant (K_d) of 12.2 nM. The maximum number of binding sites was estimated to be 17 pmol DDT/mg membrane protein (0.34 pmol/house fly head). Competition studies showed DDT, cis-permethrin, and cypermethrin bind to the same receptor but not at precisely the same site. The addition of Ca²⁺ to the incubation buffer significantly inhibited the binding of both [¹⁴C]DDT and [¹⁴C]cis-permethrin, suggesting the receptor binding is Ca²⁺ sensitive and may have a role in ion conductance.     

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.     

Species differences in chlorantraniliprole and flubendiamide insecticide binding sites in the ryanodine receptor

Anthranilic and phthalic diamides exemplified by chlorantraniliprole (Chlo) or cyantraniliprole (Cyan) and flubendiamide (Flu), respectively, are the newest major chemotype of insecticides with outstanding potency, little or no cross resistance with other classes and low mammalian toxicity. They are activators of the ryanodine (Ry) receptor (RyR)-Ca²⁺ channel, based on Ca²⁺ flux and electrophysiology investigations. The goal of this study is to define species differences in the degree and mechanisms of diamide selective action by radioligand specific binding studies at the [³H]Ry, [³H]Chlo and [³H]Flu sites. The [³H]Ry site is observed in muscle of lobster, rabbit and four insect species (Musca domestica, Apis mellifera, Heliothis virescens and Agrotis ipsilon) whereas the [³H]Chlo site is evident in the four insects and the [³H]Flu site in only the two lepidoptera (Agrotis and Heliothis). [³H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit. [³H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory. [³H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis. [³H]Chlo and [³H]Flu binding are not dependent on added Ca²⁺ or ATP in Heliothis and Agrotis whereas the other radioligand-receptor combinations are usually enhanced by Ca²⁺ and ATP. More generally, there are species differences in the Ry, Chlo and Flu binding sites of the RyR that may confer selective toxicity and determine target site cross resistance mechanisms.     

Effects of monoterpenoid insecticides on [H]-TBOB binding in house fly GABA receptor and Cl uptake in American cockroach ventral nerve cord

Monoterpenoids and their derivatives from plant essential oils showed good insecticidal activities in previous studies, but the mechanisms of their action as natural insecticides are not known yet. In the present work, we evaluated the pharmacological action of five monoterpenoids (α-terpineol, carvacrol, linalool, pulegone, and thymol) on native insect GABA receptors from house flies and American cockroaches using radiotracer methods. In the [³H]-TBOB binding assay, carvacrol, pulegone, and thymol all enhanced the [³H]-TBOB binding to membrane preparation of house fly heads with EC₅₀ values of 48 μM, 432 μM, and 6 mM, respectively. Moreover, these three monoterpenoids at concentrations of 500 μM and 1 mM also significantly increased the ³⁶Cl⁻ uptake induced by GABA in membrane microsacs prepared from American cockroach ventral nerve cords. These results revealed that carvacrol, pulegone, and thymol are all positive allosteric modulators at insect GABA receptors. The other two monoterpenoids that were tested, α-terpineol and linalool, showed little or no effect in both the [³H]-TBOB binding and ³⁶Cl⁻ uptake assays.     

Quantitative autoradiography of GABA receptors in locust (Schistocerca americana) brain

Conventional film autoradiography was used at the light microscopic level for the localization and quantization of 4-aminobutyric acid (GABA) receptors in the locust brain (Schistocerca americana). Localization of the receptor site was achieved via binding with the receptor-ligand probe [³ H]muscimol. Frozen sections were cut and subsequently incubated either in 40 nM [³H]muscimol or by coincubating sections with [³H]muscimol and one of the following: GABA (50 μM)], a receptor specific agonist [muscimol (1 μM) or isoguvacine (1 μM)], an uptake inhibitor [nipecotic acid (50 μM)], or a noncompetitive channel modulator [avermectin B_1a, (1 μM) or aldrin (50 μM)]. Through computer image enhancement and densitometric analysis of the optical density of [³H]muscimol binding sites, the interaction of the above compounds with the putative GABA receptor was determined for various anatomical regions of the locust brain. By comparing the differently treated, but adjacent sections, GABA receptor distribution was quantitated and mapped. Receptor sites were found distributed in the antennal lobes, central body, β-lobe and β-lobe of the corpus pedunculatum, protocerebral bridge, and calyx as well as the optic lobe regions.     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites

The ability of spinosyn A to either enhance or displace binding to selected insecticidally-relevant receptors was investigated using a number of radioligands including, [³H]imidacloprid and [³H]ivermectin in tissues from the ventral nerve cord (VNC) membranes of the American cockroach, Periplaneta americana and head membranes from the housefly, Musca domestica. In these insect neural tissues, spinosyn A does not appear to alter the binding of a number of radioligands suggesting that spinosyn A does not interact directly with a variety of known receptors, including nicotinic or γ-aminobutyric acid (GABA)-based insecticidal target sites. However, available data are consistent with spinosyn A interacting with a site distinct from currently known insecticidal target sites, thus supporting a novel insecticidal mechanism of action for the spinosyns.     

Pyrethroid action on the nicotinic acetylcholine receptor/channel

The interactions of natural pyrethrins and nine pyrethroids with the nicotinic acetylcholine (ACh) receptor/channel complex of Torpedo electric organ membranes were studied. None caused significant reduction in [³H]ACh binding to the receptor sites, but all inhibited [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) binding to the channel sites in presence of carbamylcholine. Allethrin inhibited [³H]H₁₂-HTX binding noncompetitively, but [³H]imipramine binding competitively, suggesting that allethrin binds to the receptor's channel sites that bind imipramine. The pyrethroids were divided into two types according to their actions: type I, which included pyrethrins, allethrin, bioallethrin, resmethrin, and tetramethrin, was more potent in inhibiting [³H]H₁₂-HTX binding and acted more rapidly (i.e., in <30 sec). Type II, which included permethrin, fluvalinate, cypermethrin and fenvalerate, was less potent and their potency increased slowly with time. Also, inhibition of the initial rate of [³H]H₁₂-HTX binding by type I compounds increased greatly by the presence of the agonist carbamylcholine, but this was not so with type II compounds. The receptor-regulated ⁴⁵Ca²⁺ flux into Torpedo microsacs was inhibited by pyrethrins and pyrethroids, suggesting that their action on this receptor function is inhibitory. There was very poor correlation between the potencies of pyrethrins and pyrethroids in inhibiting [³H]H₁₂-HTX binding and their toxicities to house flies, mosquitoes, and the American cockroach. However, the high affinities that several pyrethroids have for this nicotinic ACh receptor suggest that pyrethroids may have a synaptic site of action in addition to their well known effects on the axonal channels.     

Desnitroimidacloprid and Nicotine Binding Site in Rat Recombinant α4β2 Neuronal Nicotinic Acetylcholine Receptor

Desnitroimidacloprid (desnitro-IMI) is proposed to be a bioactivation product of imidacloprid and to bind at the same site as [³H]nicotine in the nicotinic acetylcholine receptor (nAChR) of mouse brain membranes. The α4β2 nAChR subtype accounts for >90% of the binding sites for nicotine in rat brain. This study further characterizes the binding site for [³]desnitro-IMI and [³H]nicotine in rat recombinant α4β2 nAChR using receptor expressed in Sf9 insect cells so that the assays involved no other receptor subtypes or interference from metabolic activation and detoxification systems. The two radioligands gave the same B_max of 7.5 pmol/mg protein and apparent K_d values of 3.3 nM for nicotine and 8.9 nM for desnitro-IMI by Scatchard analysis at 22°C. However, at 4°C, the observed apparent association rate is slower and dissociation rate is faster for [³H]desnitro-IMI than for [³H]nicotine and due to the rapid rate of dissociation of [³H]desnitro-IMI the K_d calculated from the determined association and dissociation rates more closely approximates 1.0 for both ligands. Eight cholinergic agents and nine nicotinoids are equipotent in displacing [³H]desnitro-IMI and [³H]nicotine, with IC₅₀ values (nM) of 0.5 for epibatidine, 1 for cytisine, 4–6 for nicotine and desnitro-IMI, 15 for acetylcholine, and 155 for imidacloprid, with an overall correlation for inhibitor potencies of _r² = 0.99 (n = 17). This correlation of binding site properties extends to [³H]nicotine in the recombinant α4β2 receptor and rat brain membranes (r² = 0.99, n = 12). Thus, desnitro-IMI and nicotine bind with high affinity to the same site in rat recombinant α4β2 neuronal nAChR. This recombinant receptor can be generated in sufficient quantities for high-throughput target site screening and structural analysis of the binding site.     

Protective effect of dimercaptosuccinic acid on methylmercury and mercuric chloride inhibition of rat brain muscarinic acetylcholine receptors

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl₂) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l-[³H]quinuclidinyl benzilate ([³H]QNB). Rat brain synaptosomal membranes were exposed to HgCl₂ and MeHg. At 1 × 10^?4M. HgCl₂ caused complete inhibition of the [³H]QNB binding. The inhibition of [³H]QNB binding by HgCl₂ was still higher than 50% at 1 × 10^?8M. MeHg caused less inhibition of [³H]QNB binding than HgCl₂. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl₂ concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl₂, the inhibition of [³H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [³H]QNB binding due to MeHg or HgCl₂ intoxication. However, DMSA was more effective in reducing HgCl₂ inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [³H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl₂ inhibition of the rat brain muscarinic receptors.     

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.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

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     

Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroach Periplaneta americana

The preparation and cholinergic properties of a subcellular fraction, enriched in pinched-off nerve-endings (synaptosomes) from the central nervous system of the cockroach Periplaneta americana, are described. The endings retained their cytoplasmic components, as shown by the presence of marker enzymes and by ultrastructural examination. A carrier-mediated, high-affinity uptake of [³H] choline into the synaptosomes was demonstrated, and this uptake was saturable, depended on a Na⁺-gradient, and was inhibited by hemicholinium-3. It had an apparent K_m value of 0.6 (±0.1) μM, and a V_max of 20.5 (±2.5) pmol min^?1 per mg of protein. The high-affinity [³H]choline uptake was associated with the synthesis of [³H]phosphocholine and [³H]O-acetylcholine. The rate of [³H]choline uptake in synaptosomes was increased by DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane] at 100 nM concentration, and this increase was inhibited by tetrodotoxin, while neostigmine appeared to be a potent inhibitor (I₅₀ = 10 pM) of the DDT-activated uptake of [³H]choline. The site of action of the insecticides was specifically on the pre-synaptic nerve terminals because the synaptosomes preparation did not retain the post-synaptic membrane of the original nerve-endings. Cockroach synaptosomes provided a useful in-vitro preparation for studying the effects of insecticides on the pre-synaptic nerve endings.     

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     

House fly head GABA-gated chloride channel: [3 H] α-Endosulfan binding in relation to polychlorocycloalkane insecticide action

This study attempts to use [³H] α-endosulfan to examine directly the binding site(s) for cyclodienes, lindane and toxaphene (collectively referred to as the polychlorocycloalkane or PCCA insecticides) in the 4-aminobutyric acid (GABA)-gated chloride channel. [³H] α-Endosulfan was prepared by reduction of hexachloronorbornenedicarboxylic anhydride with sodium borotritide, then coupling the labelled alcohol with thionyl chloride followed by HPLC purification (35 Ci mmol^?1, > 99% radiochemical purity). This new candidate radioligand readily partitions into lipid membranes and undergoes indiscriminate adsorption to surfaces resulting in high levels of non-specific binding. This makes it very difficult to differentiate the small portion of specific binding at the site relevant to toxic action. This problem was partially circumvented by incubating [³H] α-endosulfan (0.1 nM) with house-fly head membranes (0.2 mg protein) for 70 min at 22°C giving 23 (±4)% specific binding (40 fmol mg^?1 protein) determined as the difference between the radioligand alone and on preincubation for 15 min with unlabelled α-endosulfan (final concentration 100 nM). This procedure is not appropriate for determination of saturation isotherms and standard binding kinetics. However, the effectiveness of 16 PCCAs (also at 100 nM final concentration) in blocking the specific binding of [³H] α-endosulfan is generally consistent with their relative potencies as inhibitors of 4-[³H] propyl-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2] octane ([³H]EBOB) binding suggesting that the binding site for both [³H]α-endosulfan and the PCCAs is part of the GABA-gated chloride channel. Insecticidal channel blockers of other types (e.g. picrotoxinin, trioxabicyclooctanes, a dithiane, and phenylpyrazoles) and GABA are poor inhibitors of [³H] α-endosulfan binding relative to their potencies as inhibitors of [³H] EBOB binding. It therefore appears that the PCCAs compete directly for the [³H] α-endosulfan site, whereas the other channel blockers bind with different inhibition kinetics or at a site more closely coupled to the EBOB than the α-endosulfan binding domain.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

Heterocyclic Insecticides Acting at the GABA-Gated Chloride Channel: 5-Alkyl-2-arylpyrimidines and -1,3-thiazines

5-tert-Butyl-2-(4-ethynylphenyl)pyrimidine and the corresponding 2,5-disubstituted-4H-1,3-thiazine block the GABA-gated chloride channel at c.20and c.200 nm , respectively, measured as 50% inhibition of the binding of 1-(4-ethynylphenyl)-4-[³H]propyl-2,6,7-trioxabicyclo[2.2.2]octane (4′-ethynyl-4-n-[³H]propylbicycloorthobenzoate; [³H]EBOB) in house fly and mouse brain membranes, and they are also toxic to topically-treated flies with LD₅₀ values of 6–27 μg g⁻¹ alone and 2–6 μg g⁻¹ with piperonyl butoxide (PB) as synergist. In the pyrimidine series, the general pattern of effectiveness of substituents in the 5-position is tert-butyl>isopropyl≈cyclohexyl≈cyclopropyl>methyl, phenyl and 3- and 4-fluorophenyl, and in the 2-position is 4-ethynylphenyl≪4-bromophenyl. These planar pyrimidines and nearly-planar 4H-1,3-thiazines with 2-ethynylphenyl or 2-bromophenyl and 5-tert-butyl or 5-isopropyl substituents are more effective than the corresponding 6H-1,3-thiazine, 6-oxo-1,3-thiazines and 4,6-dioxo-1,3-thiazine examined, but they are less active than the analogous conformationally flexible trans-1,3-dioxanes and -1,3-dithianes. The heterocyclic moiety confers a region of high electron density and positions the 2- and 5-substituents in a linear or parallel relationship for optimal affinity at the receptor. Two observations indicate that the new pyrimidines and thiazines probably act as chloride channel blockers. First, the poisoning signs are identical to those of EBOB in both mice and house flies. Second, each of the pyrimidines, thiazines and dioxanes falls on the same correlation line for inhibition of [³H]EBOB binding and toxicity to house flies (with PB) as that obtained earlier for EBOB analogs, dithianes and polychlorocycloalkanes, suggesting that they all act at the same or closely coupled binding sites in the GABA-gated chloride channel.     

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.