Insect nAChRs

In insects, genes are identified encoding multiple uAChR subunits, suggesting the existence of diverse insect receptor subtypes. As an agonist-gated ion channel complex for rapid excitatory neurotransmission, the nAChR is widely distributed in insect CN S and constitutes a major target for insect action. However, the functional architecture and diversity of insect nAChRs are poorly understood compared with their vertebrate counterparts [91]. In general, insect nAChRs are diverse in structure, as are those from vertebrates. 

As in vertebrates, in insects nAChRs mediate fast synaptic transmission as an excitatory neurotransmitter-receptor complex widely distributed in the synaptic neutrophil regions of the CNS in the insect brain [93, 94]. 

Three further putative nAChR a subunits (Da5-Da7) with sequence similarity to the vertebrate oi7 subunit have been identified from Drosophila genome sequence data but there have been no reports, as yet, of their characterization by heterologous expression [96]. 

Generally, insect nAChRs clearly vary with specificity of their interaction with neonicotinoid insecticides however, the appropriate subunit is unclear so far. An investigation that supports the hypothesis that there is a conserved neonicotinoid special sensitive subtype of the nAChR binding site in different insects like Musca domestica, D. melanogaster, Aphis craccivora, Myzus persicae has been discussed [97]. 

Whenever it has not been possible to obtain crystals of any nAChR of sufficient quality for high-resolution X-ray crystallography, both the crystal structure of a soluble homopentameric AChBP and the refined model of the membrane-associated Torpedo AChR [98], based on the crystal structure of AChBP, can support the understanding of the ligand-receptor interactions considerably. 

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Understanding the selectivity of neonicotinoids toward insect nAChR is essential for environment protection, human health, and insecticide resistance [7], and also a key issue for the design and structure-relationship of new derivatives. 

Computational model. Based on the bioinformatic analysis and the known mutation data [8-10] of insect nAChR, two key residues (Arg and Trp) were selected to build a computational model with three analogs of neonicotinoids. Considering the computational efficiency, the structures were simplified as seen in Figure 1. 

Figure 2. Schematic representation of imidacioprid-insect nAChR interactions.

Compared to the marked effects of the double mutations in loop D of the p2 subunit on the neonicotinoid sensitivity of the a4p2 and Da2p2 nAChRs, the effects of the mutations on the concentration-response curves of ACh were minimal (Figure 6). These findings suggest that loop D offhe insect nAChR plays a role in recognizing the structural features of neonicotinoids. 

Table 29.1.2 Amino acid sequence in loop D of vertebrate and insect nAChR subunits. (Data taken from Shimomura et al., 2002.)...

II). Thus the 7t-conjugated system composed of a N-nitro-imino- or N-cyano-imino group and the conjugated nitrogen in 1-position are considered essential moieties for the binding of neonicotinoids to the putative cationic subsite in insect nAChR. 

Since the discovery of 7, diverse imidacloprid-related insecticides referred to as neonicotinoids have been synthesized. Like 7, all commercial neonicotinoids 8-13 bind with high affinity (I50 1 mM) to [ H]-7 binding sites on insect nAChRs. 

Neonicotinoid insecticides are more than 100-fold selective for insect nAChRs over vertebrate nAChRs, but little is known about the mechanism of selectivity... 

Spinosad (15) as a reduced-risk insecticide with wider margins of safety for nontarget organisms demonstrates also the different sensitivity of insect nAChRs versus mammalian nAChRs (see Chapter 29.3). 

Pharmacological profiles of the recombinant hybrid insect a/vertebrate yS nAChRs are poorly defined and the binding sites are not established for identified subunits versus native receptors. Functional expression of insect nAChRs of known subunit compositions facilitates understanding of the mechanism underlying these molecular interactions. However, it is difficult to heterologously express functionally robust nAChRs not only in Xenopus oocytes but also in Drosophila S2 cells [186]. Only a few functional receptors have been obtained after expression of different subunit combinations in X. oocytes or cell lines. Nevertheless, Drosophila nAChR a subunits can form homo-oligomeric functional nAChRs when coexpressed with a vertebrate pi (non-a) subunit in X. oocytes. This has been demonstrated so far for ... 

These examples indicate the complexity of insect nAChRs, which is difficult to understand. The considerable diversity of potential subunit combinations probably accounts for the multiplicity of distinctive pharmacological profiles in insect nAChRs. In that context, electrophysiology will play an essential role in determining the significance of certain subunit combinations in the MoA of neonicotinoid and further insecticidally active ligands. 

Table 29.1.4 Comparison between electro-physiological and [ H]-7 displacement potencies for different neonicotinoids 6-9, 13 and ( )-epibatidin (16) on insect nAChRs. Electrophysiological data [ECso and relative (agonist) efficacy] were obtained from neuron cell bodies isolated from the CNS of H. virescens. EC50 and relative efficacy values represent the mean of separate experiments on different neurons. Inhibition of [ H]-7 binding to nAChR in housefly head membrane preparations by the compounds is expressed as piso (piso values (= -log M) correspond to the concentration of cold ligand displacing 50% of bound [ H]-7 from housefly head membranes).

The selectivity of neonicotmoids for insect over vertebrate nAChRs is likely to result from selective recognition by insect uAChRs of its structural features and vice versa. To elucidate the mechanism of selectivity, structural features of neonicoti-noids and insect nAChRs contributing to this selectivity have been examined. 

Fig. 29.1.5. Binding subsite specificity shown as hypothetical schematic models for the CNI imidacloprid (7) binding in the insect nAChR and nicotinoid /V-cfes-nitro-7 binding in the mammalian nAChR, each at the ACh agonist site (Adapted from Tomizawa and Casida, 2005 [169]).

There are numerous examples of isosterism between open-chain and ring systems among bioactive molecules [68]. In comparison to the corresponding five-and six-membered ring systems (Chapters 29.2.2 and 29.2.3, respectively), the open-chain compounds exhibit similar broad insecticidal activity, forming a so-called quasi-cyclic conformation when binding to the insect nAChR [69]. 

Neonicotinoids are insecticides acting on insect nAChRs (1). For a long time they are very effective on commercially important Hemipteran pest species such as aphids, whiteflies and planthoppers, but also control Coleopteran and some Lepidopteran pests (/). The biochemical mode of action (MoA) of neonicotinoids has been studied and characterized extensively in the past 10 years. Ail neonicotinoids act selectively as agonists at the insect nAChRs and they are part of a single MoA group as defmed by the Insecticide Resistance Action Committee (IRAC an Expert Conunittee of Crop Life) for resistance management purposes (2). Today the neonicotinoids are ... 

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