Cholinergic Insecticide Toxicology

描述（由申请人提供）：新烟碱类杀虫剂是过去三十年中最重要的一类新型杀虫剂，已占世界杀虫剂市场的10%左右，人类暴露量很大。它们具有与尼古丁相同的尼古丁乙酰胆碱受体（nAChR）靶标，但对昆虫的作用更强，选择性更强。长期目标是确定主要新烟碱类、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新烟碱和烟碱结构特征。带负电荷的尖端被认为赋予昆虫nachr的效力和选择性，促使我们合成具有这一特征的结构探针。优选的构象和构型将由x射线晶体学和核磁共振确定，并与效力有关。量子力学研究将建立静电势面、分子电荷分布和结合能。同样的方法将用于表征哺乳动物nachr选择性的类似烟碱。第二个目的是表征决定选择性作用的nAChR亚型、亚基和亚位点。一个目标是确定alpha4beta2， alpha3beta2（和/或beta4） alpha5，或alpha7和alpha1Gamma alpha1deltabeta1亚型在结合新烟碱及其代谢物中的作用，相对于功能测定和选择性毒性。将果蝇nAChR和脊椎动物nAChR亚型溶解，用新烟碱或烟碱亲和层析纯化，并用本实验室设计的强效叠氮酮烟碱和叠氮酮烟碱光亲和探针进行标记。标记的亚基将被识别，特别注意昆虫中的阳离子亚基和脊椎动物中的pi-富电子亚基。详细结合位点结构的分子建模将把果蝇d - α亚基和乙酰胆碱结合蛋白的结构和光亲和标记的发现联系起来。第三个目的是确定新烟碱代谢激活和解毒相对于选择性毒性。将对吡虫啉、噻虫啉、噻虫嗪和啶虫啉的代谢物进行表征和合成，用于受体、毒性和功能分析，以阐明选择性代谢激活与解毒。对小鼠进行的毒物动力学研究将把新烟碱类及其二硝基和二硝基代谢物（有毒的亚胺衍生物）的大脑水平与中毒症状联系起来。继续的研究将确定人类细胞色素P450同功酶在新烟碱类代谢中的特异性，并表征人类微粒体“新烟碱类硝基亚胺还原酶”，该酶产生独特的吡虫啉腙和三唑酮衍生物。

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.