Cholinergic Insecticide Toxicology

描述（由申请人提供）：新烟碱类杀虫剂是过去三十年来最重要的新型杀虫剂，已占世界杀虫剂市场的约10%，并且主要是人类暴露。它们具有与尼古丁相同的烟碱乙酰胆碱受体（nAChR）靶点，但对昆虫更有效和更有选择性。长期目标是确定主要烟碱类杀虫剂、吡虫啉、噻虫啉、噻虫嗪和啶虫脒的选择性毒性机制。第一个具体目标是建立nAChR特异性和选择性毒性的独特的类烟碱和烟碱结构特征。负电荷的提示，建议赋予昆虫nAChRs的效力和选择性，促使我们合成具有此功能的结构探针。优选的构象和构型将通过X射线晶体学和NMR确定，并与效价相关。量子力学的研究将建立静电势面、分子电荷分布和结合能。将使用相同的方法来表征对哺乳动物nAChR具有选择性的类似烟碱。第二个目的是表征nAChR亚型，亚基和亚位点，决定选择性行动。一个目标是确定α 4 β 2、α 3 β 2（和/或β 4）、α 5或α 7和α 1 γ α 1 δ β 1亚型在结合类尼古丁及其代谢物中相对于功能测定和选择性毒性的作用。果蝇nAChR和脊椎动物nAChR亚型溶解，纯化的类烟碱或烟碱亲和层析，并与本实验室设计的有效的azidoneonicotinoid和azidonicotinoid光亲和探针标记。标记的亚基将特别注意昆虫中的阳离子亚位点和脊椎动物中的富π电子亚位点。详细的结合位点结构的分子建模，然后将果蝇D-α亚基和乙酰胆碱结合蛋白的结构与光亲和标记的结果。第三个目的是定义与选择性毒性相关的类烟碱代谢活化和解毒。将对吡虫啉、噻虫啉、噻虫嗪和啶虫脒的代谢产物进行表征和合成，用于受体、毒性和功能测定，以澄清选择性代谢活化与解毒。小鼠毒代动力学研究将使类烟碱及其去硝基和去氰基代谢物（毒性亚胺衍生物）的脑水平与中毒体征相关。继续研究将确定人细胞色素P450同工酶在类烟碱代谢中的特异性，并表征人微粒体“类烟碱硝基亚胺还原酶”，该还原酶产生独特的腙和三唑酮衍生物吡虫啉。

DESCRIPTION (provided by applicant): Neonicotinoids are the most important new class of insecticides of the last three decades, already accounting for about 10% of the world insecticide market and with major human exposure. They have the same nicotinic acetylcholine receptor (nAChR) target as nicotine but are much more potent and selective for insects. The long term objective is to define the mechanisms of selective toxicity for the major neonicotinoids, imidacloprid, thiacloprid, thiamethoxam and acetamiprid. The first specific aim is to establish the unique neonicotinoid and nicotinoid structural features for nAChR specificity and selective toxicity. The negatively-charged tip is proposed to confer potency and selectivity for insect nAChRs, prompting us to synthesize structural probes with this feature. The preferred conformation and configuration will be determined by X-ray crystallography and NMR and related to potency. Quantum mechanics studies will establish the electrostatic potential surface, molecular charge distribution and binding energy. The same approaches will be used to characterize the analogous nicotinoids selective for mammalian nAChRs. The second aim is to characterize nAChR subtypes, subunits and subsites that determine selective action. One goal is to establish the role(s) of alpha4beta2, alpha3beta2(and/or beta4) alpha5, or alpha7 and alpha1Gamma alpha1deltabeta1 subtypes in binding neonicotinoids and their metabolites relative to functional assays and selective toxicity. Drosophila nAChR and vertebrate nAChR subtypes are solubilized, purified by neonicotinoid- or nicotinoid-affinity chromatography, and labeled with potent azidoneonicotinoid and azidonicotinoid photoaffinity probes designed in this laboratory. The labeled subunits will be identified with particular attention to the proposed cationic subsite(s) in insects and pi-electron-rich subsite(s) in vertebrates. Molecular modeling of the detailed binding site architecture will then relate the structure of the Drosophila D-alpha subunits and the acetylcholine binding protein to the findings on photoaffinity labeling. The third aim is to define neonicotinoid metabolic activation and detoxification relative to selective toxicity. Metabolites of imidacloprid, thiacloprid, thiamethoxam and acetamiprid will be characterized and synthesized for receptor, toxicity and functional assays to clarify selective metabolic activation versus detoxification. Toxicokinetic studies with mice will relate the brain levels of neonicotinoids and their desnitro and descyano metabolites (toxic iminium derivatives) to the poisoning signs. Continuing investigations will define human cytochrome P450 isozyme specificity in metabolism of neonicotinoids and characterize human microsomal "neonicotinoid nitroimine reductase" that generates unique hydrazone and triazolone derivatives of imidacloprid.