Miki Akamatsu

Neonicotinoids Show Selective and Diverse Actions on Their Nicotinic Receptor Targets: Electrophysiology, Molecular Biology, and Receptor Modeling Studies

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin.

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs.

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken α4β2 nAChR and Drosophila melanogaster Dα2/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the β2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the α4β2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dα2β2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant α4β2 and Dα2β2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Diverse actions of neonicotinoids on chicken α7, α4β2 and drosophila-chicken SADβ2 and ALSβ2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes

The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the alpha7 receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor.

Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists

A CXCR4 antagonistic peptide, T140, and its bio-stable analogs, such as Ac-TE14011, were previously developed. These peptides inhibit the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into T cells. Herein, a series of TE14011 analogs having modifications in the N-terminal region were synthesized to develop effective compounds with increased biostability. Among these analogs, 4F-benzoyl-TE14011 (TF14013) showed the strongest anti-HIV activity derived from CXCR4-antagonism, suggesting that a 4-fluorobenzoyl moiety at the N-terminus of T140 analogs constitutes a novel T140-based pharmacophore for CXCR4 antagonists. Structure-activity relationship (SAR) studies on TE14011 analogs with N(alpha)-acylation by several benzoic acid derivatives have disclosed a significant relationship between the anti-HIV activity and the Hammett constant (sigma) of substituted benzoic acids. TF14013 was found to be stable in mouse serum, but not completely stable in rat liver homogenate due to deletion of the C-terminal Arg14-NH2 from the parent peptide. This biodegradation was completely suppressed by N-alkyl-amidation at the C-terminus. Taken together, the enhancement of the T140-based pharmacophores led to development of a novel CXCR4 antagonist, 4F-benzoyl-TE14011-Me (TF14013-Me), which has very high anti-HIV activity and increased biostability.

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits.

Effects of mutations of a glutamine residue in loop D of the α7 nicotinic acetylcholine receptor on agonist profiles for neonicotinoid insecticides and related ligands

Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site‐directed mutagenesis of the chicken α7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP). In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that corresponds to G189 of the α7 nicotinic receptor. Since Q55 of AChBP is preserved as Q79 in the α7 nicotinic receptor and the G189D and G189E mutations have been found to reduce the neonicotinoid sensitivity, we investigated effects of Q79E, Q79K and Q79R mutations on the neonicotinoid sensitivity of the α7 receptor expressed in Xenopus laevis oocytes to evaluate contributions of the glutamine residue to nicotinic AChR–neonicotinoid interactions. The Q79E mutation markedly reduced neonicotinoid sensitivity of the α7 nicotinic AChR whereas the Q79K and Q79R mutations increased sensitivity, suggesting electronic interactions of the neonicotinoids with the added residues. By contrast, the Q79E mutation scarcely influenced responses of the α7 nicotinic receptor to ACh, (−)‐nicotine and desnitro–imidacloprid (DN–IMI), an imidacloprid derivative lacking the nitro group, whereas the Q79K and Q79R mutations reduced the sensitivity to these ligands. The results indicate that the glutamine residue of the α7 nicotinic receptor is likely to be located close to the nitro group of the insecticides in the nicotinic receptor–insecticide complex.

Prediction of the binding mode of imidacloprid and related compounds to house-fly head acetylcholine receptors using three-dimensional QSAR analysis

The binding activity of imidacloprid and related compounds to nico- tinic acetylcholine receptors (nAChR) of house Nies was measured by use of radioactive a-bungarotoxin as a ligand. Variations in the activity were examined three-dimensionally using comparative molecular -eld analysis (CoMFA). The CoMFA results suggest that one conformer among the four stable ones is active and provide support for one of the proposed binding models for this class of compound, in which the nitrogen atom of the pyridine ring and the nitrogen atom at the 1-position of the imidazolidine ring interact with the hydrogen- donating and electron-rich sites of nAChR, respectively. The CoMFA -eld map showed that the nitroimino moiety and a portion of the imidazolidine ring were mainly surrounded by a sterically and electrostatically sensitive region of nAChR. 1998 Society of Chemical Industry ( Pestic. Sci., 54, 134E144 (1998)

Comparative pharmacology and computational modelling yield insights into allosteric modulation of human α7 nicotinic acetylcholine receptors

The human alpha7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type II PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type II PAM, PNU-120596, are all active on human alpha7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders.

Three-dimensional quantitative structure: activity relationship analysis of acyclic and cyclic chloronicotinyl insecticides

The binding activity of chloronicotinyl insecticides, including acetamiprid, nitenpyram and related compounds, to the nicotinic acetylcholine receptors (nAChR) of houseflies was measured. These compounds were defined as ‘acyclic’ compounds. Variations in the binding activity were analysed using comparative molecular field analysis (CoMFA) which is a technique for the analysis of three-dimensional quantitative structure–activity relationships. The CoMFA results showed that steric interactions were more significant for the acyclic compounds than for imidacloprid and its derivatives (cyclic compounds). It was also shown that the acyclic compounds could bind to housefly-nAChR in a similar manner to the cyclic compounds, and that the electrostatic natures of the acyclic amino- and cyclic imdazolidine-moieties affected their binding activity. © 2000 Society of Chemical Industry