Mapping the elusive neonicotinoid binding site

Abstract

Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid and thiacloprid, are nonprotonated and selective for insects and their nicotinic receptors, whereas nicotinoids such as nicotine and epibatidine are cationic and selective for mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), as a structural surrogate for the extracellular ligand-binding domain of the nicotinic receptor, is similarly sensitive to neonicotinoids and nicotinoids. It therefore seemed possible that the proposed very different interactions of the neonicotinoids and nicotinoids might be examined with a single AChBP by using optimized azidochloropyridinyl photoaffinity probes. Two azidoneonicotinoids with a nitro or cyano group were compared with the corresponding desnitro or descyano azidonicotinoids. acetylcholine binding protein ͉ imidacloprid ͉ neonicotinoid insecticides ͉ nicotinic receptor ͉ photoaffinity labeling S elective toxicity is critical for insecticide use, combining outstanding effectiveness for pests with safety for humans and wildlife. Neonicotinoids, exemplified by the major imidacloprid (IMI), thiacloprid (THIA), and acetamiprid (ACE) (Figs. 1 and 2), are the most important new class of insecticides of the past three decades and are increasingly replacing the organophosphates and methylcarbamates. The favorable selectivity of the neonicotinoids occurs largely at the target level, which is the agonist binding site of the nicotinic acetylcholine (ACh) receptor (nAChR). Nicotine, the namesake of the nAChR, has been used for pest control since the 17th century despite the risk for people and limited insecticidal efficacy. In contrast, the new and nicotine-like neonicotinoids have low activity on mammalian relative to insect nAChRs, providing a mechanistic basis for their safety (1, 2).Primary insights into the structure and function of agonistgated ion channel complexes came from electron microscopy analysis of the Torpedo nAChR (3) and x-ray crystallography of ACh binding proteins (AChBPs), soluble homologues of the nAChR extracellular drug-binding domain (4-7). Nicotine and its more potent analog epibatidine (EPI) (Fig. 2) (8) played a major role in structural characterization of the agonist-binding domain. Protonation of these agonists at physiological pH is important for binding site interactions at the superfamily of neurotransmitter-gated ion channels (5, 7, 9-11). Neonicotinoids, on the other hand, are uncharged with an electronegative nitro or cyano pharmacophore (12, 13). The first step in understanding neonicotinoid binding sites was identification of the molecular determinants for target site resistance and sitedirected mutagenesis (14-16), but such studies do not provide a complete description of the binding site.We report here that AChBP from the saltwater mollusk Aplysia californica surprisingly shows high sensitivity to the neonicotinoids. Photoaffinity labelin...

Structure-guided drug design: Conferring selectivity among neuronal nicotinic receptor and acetylcholine-binding protein subtypes

“…Nicotinoids and Neonicotinoids-These compounds present an interesting series owing to their selectivity for the insect receptor and their commercial applications as insecticides [4,5]. Radiolabeling experiments show that these ligands bind to the agonist site and with the pyridine ring system positioned similar to that in epibatidine or nicotine [4,5].…”

“…Radiolabeling experiments show that these ligands bind to the agonist site and with the pyridine ring system positioned similar to that in epibatidine or nicotine [4,5]. The unique characteristics of the neonicotinoids stem from substitution of the cationic nitrogen of the tertiary or quaternary agonists that stimulate the mammalian receptor.…”

“…The unique characteristics of the neonicotinoids stem from substitution of the cationic nitrogen of the tertiary or quaternary agonists that stimulate the mammalian receptor. The substitution renders the neonicotinoid nitrogen position neutral to anionic [4,5].…”

“…Moreover, if we extend our considerations beyond the mammalian species, we see even greater structural diversity, and the determinants governing ligand recognition between insect and mammalian nicotinic receptors are the basis for the selective toxicity of the neonicotinoids enjoying current use as insecticides [4,5] Of the nine subunits in neuronal nicotinic receptors in mammals, designated as α2 through α10 and containing a vicinal disulfide on the C loop of their principal binding face, α2, α3, α4 and α6 associate with the complementary β2, β3 and β4 subunits to form pentameric molecules. The α5 subunit behaves similarly to a β subunit in not having its principal face creating a binding site.…”

“…Since the nicotinic receptor in skeletal muscle forms the signal transducer for neuromuscular transmission controlling motor activity, and in the CNS, nicotinic receptors primarily mediate release of neurotransmitters from their presynaptic sites, a variety of toxins from marine snails, terrestrial plants, coral as stationary animals, and reptiles have evolved to block or modulate nicotinic receptor function. Several of these compounds, in contrast to acetylcholine, carbamylcholine, nicotine and epibatidine, show considerable selectivity towards receptor subtype [1][2][3][4][5]. Therefore these compounds can be considered as lead templates for developing selective ligands.…”