James J. Rauh

Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors

Imidacloprid is increasingly used worldwide as an insecticide. It is an agonist at nicotinic acetylcholine receptors (nAChRs) and shows selective toxicity for insects over vertebrates. Recent studies using binding assays, molecular biology and electrophysiology suggest that both alpha- and non-alpha-subunits of nAChRs contribute to interactions of these receptors with imidacloprid. Electrostatic interactions of the nitroimine group and bridgehead nitrogen in imidacloprid with particular nAChR amino acid residues are likely to have key roles in determining the selective toxicity of imidacloprid. Chemical calculation of atomic charges of the insecticide molecule and a site-directed mutagenesis study support this hypothesis.

Ion channels: molecular targets of neuroactive insecticides

Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p′-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); γ-aminobutyric acid (GABA) receptors (cyclodienes, γ-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.

Pharmacological and biochemical properties of insect GABA receptors

The first evidence for the existence of GABA receptors in any tissue was provided by studies on an invertebrate preparation but, until recently, characterization of GABA receptors from such lower organisms has advanced slowly. The identification of GABA receptors as putative target sites for a variety of insecticidal agents has contributed to the resurgence of interest in amino acid receptors of insects and other invertebrates. In this review, James Rauh and colleagues describe the properties of GABA receptors of insects and detail some striking pharmacological differences between the well-characterized GABA receptors of vertebrates and those of insects and other invertebrate organisms. A detailed understanding of invertebrate receptor pharmacology will be increasingly important for defining the mode of action of numerous modern pesticides.

Pharmacology of insect GABA receptors

A GABA-operated Cl− channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl− channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABAA receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl− channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.

Effects of [3H]‐BIDN, a novel bicyclic dinitrile radioligand for GABA‐gated chloride channels of insects and vertebrates

The radiolabelled bicyclic dinitrile, [3H]‐3,3‐bis‐trifluoromethyl‐bicyclo[2.2.1]heptane‐2,2‐dicarbonitrile ([3H]‐BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ‐aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35±3 nM) and dieldrin (IC50, 93±7 nM), displaced [3H]‐BIDN from rootworm membranes. When tested at 100 μM, 1‐(4‐ethynylphenyl)‐4‐n‐propyl‐2,6,7‐trioxabicyclo[2.2.2]octane(EBOB), 4‐t‐butyl‐2,6,7‐trioxa‐1‐phosphabicyclo[2.2.2]octane‐1‐thione (TBPS), 1‐phenyl‐4‐t‐butyl‐2,6,7‐trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]‐BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [3H]‐BIDN from rootworm membranes by a competitive mechanism. Rat brain membranes were also shown to possess a population of saturable, specific [3H]‐BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]‐BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. Functional GABA‐gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABAB receptors were unaffected. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA‐derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo‐oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA‐gated Cl− channels to which a number of insecticidally‐active molecules bind.

In vitro neuropharmacological evaluation of piperovatine, an isobutylamide from Piper piscatorum (Piperaceae)

Piperovatine, a sialogogic, piscicidal, and buccal local anesthesia producing isobutyl amide from the amazonian piscicidal and toothache-relieving plant, Piper piscatorum Trelease et Yuncker (Piperaceae), was evaluated for its ability to induce changes in neuronal intracellular calcium concentration. Ratiometric calcium imaging of Periplaneta americana neuronal cell cultures upon piperovatine application revealed that this compound induced dramatic increases in intracellular calcium concentration. Calcium flux was not affected by co-application of the muscarinic acetylcholine receptor antagonist, atropine, indicating that the parasympathomimetic system was not involved in piperovatines sialogogic actions. Calcium flux was, however, totally eliminated by co-application of the voltage-gated sodium channel blocker, tetrodotoxin (TTX). This, in conjunction with the repetitive calcium spikes observed in the assay and previous radioligand binding studies on the chemical class, strongly suggest that activation of voltage-gated sodium channels characterizes piperovatines mode of action

Tricyclic cyanoguanidines: synthesis, site of action and insecticidal activity of a novel class of reversible acetylcholinesterase inhibitors

Bridged-tricyclic cyanoguanidines 1 were found to be active as insecticides. The preparation and structure-activity relationships of oxacyclic (X=O) and carbocyclic (X=CH(2)) analogues of 1 is described. Compounds 1 were found to inhibit acetylcholinesterase with IC(50) values comparable to the organophosphate Paraoxon. Unlike organophosphates, cyanoguanidines 1 were shown to reversibly bind acetylcholinesterase. This mode of action is shared by the structurally-related natural product Huperzine A.

Blocking actions of BIDN, a bicyclic dinitrile convulsant compound, on wild-type and dieldrin-resistant GABA receptor homo-oligomers of Drosophila melanogaster expressed in Xenopus oocytes

The receptor antagonist actions are described for a novel bicyclic dinitrile compound (BIDN, 3,3-bis-(trifluoromethyl)-bicyclo [2.2.1] heptane-2,2-dicarbonitrile) on a Drosophila melanogaster homo-oligomeric GABA receptor expressed in Xenopus oocytes. BIDN blocked the wild-type form of the receptor in a neither purely competitive, nor purely non-competitive manner, being dependent on the GABA concentration yet insurmountable, and block was independent of the membrane potential. BIDN was found to be less effective against a mutant (A(302) --> S) form of the receptor resistant to dieldrin and picrotoxinin. This cross resistance of dieldrin-resistant receptors to BIDN is of interest in the light of recent findings that BIDN binding to insect membranes is displaced competitively by dieldrin, but not by picrotoxinin.

Blocking Actions of Heptachlor at an Insect Central Nervous System GABA Receptor

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range —75 mV to —110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.

Polycyclic dinitriles: a novel class of potent GABAergic insecticides provides a new radioligand, [3H]BIDN

The polycyclic dinitriles are a potent class of insecticides which are non-competitive GABA (γ-aminobutyric a acid) antagonists acting at the convulsant site. Comparison with other classes of GABA convulsant site ligands using molecular modelling has shown significant structural similarities. We have developed a pharmacophore model which unifies this class and some previous classes of GABA convulsants. Key pharmacophore elements are a polarizable functionality separated by a fixed distance from two H-bond accepting elements. This model is based on information from X-ray crystal structures and Sybyl using the Tripos force field. Using this pharmacophore model, numerous structural modifications were explored to enhance understanding of structure-activity relationships at the GABA receptor convulsant site of insects and mammals. A radiolabelled bicyclic dinitrile, [3H]BIDN ([3H]3,3-bis-trifluoromethyl-bicyclo[2,2,1] heptane-2,2-dicarbonitrile), was prepared from this area of chemistry and was used as a probe for the interaction of polycyclic dinitriles at the target site.