Binding imidacloprid

In a recent study, resistance to the neonicotinoid insecticide, imidacloprid, in the brown plant hopper, Nilaparvata lugens, was found to be due to a point mutation at a conserved position in two nAChR subunits. As a result, it reduced the receptor binding of imidacloprid (Liu et al., 2005). 

Liu M-Y and Casida JE (1993) High affinity binding of [ H] Imidacloprid in the insect acetylcholine receptor. Pesticide Biochemistry and Physiology 46 40—46. 

The toxicokinetics of the thianicotinyl thiamethoxam is similar to that of imidacloprid. When applied orally to rats, goats, or chickens, thiamethoxam is rapidly and almost quantitatively absorbed. Its excretion, predominantly in urine, is fast. Accumulation in tissues is negligible. Thiamethoxam itself does not bind strongly to the neonicotinoid binding site of the nicotinic acetylcholine receptor but it is reported to be converted to clothianidin, a neonicotinoid with high affinity for the insect receptors, in insects and plants (Figure 6). It is possible that this activation proceeds via formation of an N-desmethyl thiamethoxam intermediate, another... 

Figure 4. Homology model of the wild-type and T77R R79V double mutant of the chicken a4p2 nAChR agonist binding site with imidacloprid bound. Reproduced from reference [21] with permission of American Society for Pharmacology and Experimental Therapeutics.

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. 

Comparative binding studies indicate that imidacloprid (7) and related neonicotinoids have little or no affinity for several mammalian nAChRs. Electrophysio-logical measurements reported in numerous studies have revealed that nAChRs are widely expressed in the insect CNS on both post- and presynaptic nerve terminals, on the cell bodies of inter-, motor- and sensory neurons [170, 171, 172, 173, 174]. 

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]).

Generally, the open-chain neonicotinoids are less lipophilic than the corresponding neonicotinoids with a ring structure (Chapters 29.2.2 and 29.2.3). Based on CoMFA results, a binding model for imidacloprid (11) has been described. 

Fig. 29.2.1.1. Stable conformations of open-chain neonicotinoids (1,2), the five-membered ring system imidacloprid (11) and predicted properties of their binding site (Okazawa et al., 2000) [72].

Although the structure of flonicamid (12) has some similarity to the neonicotinoids it does not bind to the nicotinic acetylcholine receptor as directly compared with nicotine and imidacloprid [37, 53]. However, flonicamid (12) is active on the A-type potassium channel currents. The current hypothesis is that flonicamid blockade of the A-type potassium channel in the presynaptic terminal underlies its lethal effect in insects. The loss of the A-type potassium rectifying current would lead to the disruption of controlled neurotransmitter release [53]. 

Biochemical experiments with several neonicotinoids on insect membranes showed that both, thiamethoxam and imidacloprid bind to the nicotinic acetylcholine receptor. Imidacloprid however inhibits the binding of thiamethoxam, while not competing for the same binding site. Thiamethoxam and other equally non-competitive neonicotinoids, which only served as research tools, share as a common structural element the N-methyl group at position 5 of the 1,3,5-oxadiazinane ring. 

Neonicotinoid insecticides act agonistically on insect nicotinic acetylcholine receptors (nAChRs). Like imidacloprid (IMI), all neonicotinoids bind with high affinity (Iso-values 1 nM) to binding sites on... 

Neonicotinoids do not act as a homogenous class of insecticides. Radioligand receptor binding assays revealed two classes of neonicotinoids described here as competitive and non-competitive , respectively, relative to [ H]imidacloprid. Differences in affinity, mode of displacement, number of binding sites and temperature sensitivity suggest that thiamethoxam binds in a way unique among the commercial neonicotinoids. Metabolic transformation is not relevant for its insecticidal effects. 

Binding of [ H]imidacloprid to membrane preparations from whole aphids has been studied to determine its affinity and number of binding sites 8, 15). The affinity (ATj) of imidacloprid was 1.7 nM and 6.1 nM with Myzus persicae and Aphis craccivora, respectively. The binding capacities (Bbhk) were 915 finol/mg protein and 1330 taol/mg protein, respectively. 

Binding experiments revealed unusual properties of thiamethoxam at the target site (77). The optimal assay conditions to demonstrate thiamethoxam binding to aphid membranes are different and more strictly deflned than those for imidacloprid. Highest specific binding of thiamethoxam is observed at low assay temperature (2 C) with freshly prep ed membranes. Furthermore, preparations stored frozen prior to the assay are as good as fresh ones for studies with imidacloprid but not with thiamethoxam. 

At temperatures k20°C, binding capacity for thiamethoxam was significantly lower by 40-60% compared to 2°C. This was typically not the case with imidacloprid though variation was observed. It should be noted that non-specific binding was not affected by temperature with both compounds. 

For A. craccivora, specific binding of [ H]thiamethoxam was similarly sensitive to temperature and membrane quality as in M. persicae IT). The data for affinity and capacity at 2 C were about 90 nM and 1000 finol/mg protein. Higher values for A. craccivora compared to M. persicae were also obtained with imidacloprid 8, IS). 

In conclusion, temperature affects binding of thiamethoxam as well as imidacloprid in a reversible way, though the former compound is clearly stronger affected. This also suggests that the binding modes or sites of the two neonicotinoids at the target receptor are obviously not identical. 

Thiamethoxam Like Imidacloprid, Binds to a Nicotinic Receptor Site... 

To obtain conclusive evidence of the interpretation of the results from receptor binding studies, as summarized above, fonctional studies of nicotinic receptors by electrophysiological techniques would be essential. Such studies have mainly been performed with imidacloprid, as it was available first, using established lab model insects such as cockroaches or other non-target insects, lepidopteran larvae, for example (5, 6, 19). Comparable studies with receptors from true target pests (sucking insects) have yet to be performed they are likely hampered by technical difficulties related to their small size. 

Like other insecticides, thiamethoxam is transformed in the insect, crop, soil and other compartments to variable degrees to yield products that may not or may be active in their own right. An example for the latter case m the neonicotinoid class is imidacloprid, which is metabolized via hydroxylated intermediates to an olefin product that is more active than the parent compound by one order of magnitude in aphids screens and in receptor binding 20. 21). 

Neonicotinoids target nicotinic acetylcholine receptors as is mostly known from studies with imidacloprid and conveniently sized lab model insects real target pests have not yet been used for technical reasons. Hence, conclusions, especially those on receptor sensitivity, should be taken with care. On the other hand, radioligand binding studies, which usually do not impose technical hurdles, have been widely performed with neonicotinoids using membranes from target and non-target insects. 

Our studies with aphids clearly suggest that thiamethoxam, like the other examined neonicotoinoids, binds to nicotinic receptors. However, there are clear differences to the other commercial neonicotinoids as documented by a kinetic analysis of competition experiments. While thiamethoxam binds to receptors with nanomolar affinity, micromolar concentrations are required to displace imidacloprid. Further, the interaction between the two compounds is noncompetitive meaning that binding of thiamethoxam reduces the binding capacity of the receptor preparation for imidacloprid but not its affinity. Thiamethoxam shares this unusual mode of inhibition with other neonicotinoids (not commercialized) also featured by an W-Methyl group in die pharmacophore. In the competitive mode, displayed by the other commercial neonicotinoids, the capacity is unchanged, while the affinity is reduced. 

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