细胞色素氧化酶CYP3A4催化吡虫啉代谢机理的理论研究

Imidacloprid (IMI) is the first neonicotinoid insecticide which has been developed successfully. Because of its similar to nicotine, the toxicity evaluation of IMI on the human body has become the focus of attention by domestic and overseas scholars. In view that the elucidation of metabolic mechanism of IMI in the human body is beneficial for the comprehensive understanding of pharmacokinetic characteristics of IMI, this project intends to employ the quantum chemistry and the hybrid quantum mechanical/molecular mechanical (QM/MM) approach to study the metabolic mechanism of IMI catalyzed by cytochrome CYP3A4. Based on the X-ray crystallographic structure of CYP3A4 enzyme, a active center model of enzyme-catalyzed reaction is constructed. For each possible pathway, the geometries of all transition states are optimized to obtain the minimum-energy pathway. The free energy barriers are calculated to study the rate-controlling step, and to obtain the most favorable reaction pathway. The law of interaction between the substrate and enzyme is summarized. On this basis, the metabolic mechanism catalytzed by CYP3A4 enzyme with a intact structure is explored to study the role of enzyme in the metabolic process. This project’s results will compare with those in metabolic pathway 2 of IMI to clarify the optimized metabolic mechanism as well as the characteristics and differences in different metabolic pathways. In addition, our results will compare with the metabolic mechanism of nicotine to obtain the correlation between IMI and its parent compound in the metabolic process. This research will facilitate to reveal the essence of the metabolic reaction of IMI and the residual behavior of IMI in the human body, and may provide theoretical basis for toxicity warning and safe applications of IMI.

吡虫啉（IMI）是第一个成功开发的新烟碱类杀虫剂。类似于烟碱的性质使IMI对人体的毒性评价成为国内外学者关注的重点。鉴于阐明IMI在人体内的代谢机理有利于全面认识IMI的药代特征，本项目拟利用量子化学和混合量子力学/分子力学（QM/MM）方法，探索细胞色素氧化酶CYP3A4催化IMI代谢的机理。基于酶的晶体结构建立酶活性中心模型，对催化反应各条可能通道优化反应过渡态，寻找最低能量途径；计算其自由能能垒，研究其速控步骤，获得其最优反应通道，总结底物和酶的相互作用规律。在此基础上，探索完整酶环境下的催化代谢机理，研究酶在代谢过程中的作用。本项目的结果将与IMI代谢途经2作比较，以期阐明其优势机理和不同代谢途径的特征及差异。同时将与烟碱的代谢途径作比较，有望获取IMI与母体化合物在代谢过程中的联系。本项目将有助于揭示IMI在人体内的代谢行为和残留动态，可为IMI的毒性预警和安全应用提供依据。

吡虫啉（IMI）是第一个成功开发的新烟碱类杀虫剂。类似于烟碱的性质使IMI对人体的毒性评价成为国内外学者关注的重点。据报道，IMI对人体的毒性与IMI的代谢机理和代谢物性质密切相关。鉴于此，本项目利用量子化学和混合量子力学/分子力学（QM/MM）方法，系统地考察了细胞色素氧化酶CYP3A4催化IMI代谢的机理。采用分子对接和分子动力学（MD）模拟获取了IMI在CYP3A4经典血红素位点的结合模式，揭示出6个关键氨基酸残基Arg192、Phe195、Ile349、Ala285、Phe284和Phe88在酶与底物结合中所起的作用。采用QM/MM计算了酶与底物结合过程的结合自由能，指出Arg192残基作为主要作用力，通过其极性和氢键作用稳定了CYP3A4-IMI复合物的结构。设计了反式羟化（pathway 1）和顺式羟化（pathway 2）两条可能的代谢途径，考察了血红素Fe(III)中心的四重态和二重态两种自旋状态。采用拉伸QM/MM分子动力学模拟在B3LYP/6-31+G(d):ff14SB水平上研究了代谢过程的微观机理，并采用分层的ONIOM QM/MM方法在B3LYP/6-311+G(2d,2p):ff99SB//B3LYP/6-31+G(d):ff14SB和M06-2X/6-311+G(2d,2p):ff99SB//B3LYP/6-31+G(d):ff14SB水平上进行了单点能计算。结果表明，低自旋二重态的自由能垒比高自旋四重态的低，代谢反应主要发生在低自旋的二重态下。pathway 1和pathway 2都是分步反应途径。能量数据显示pathway 1的自由能垒比pathway 2低10—15 kcal/mol，揭示pathway 1反应发生的可能性较pathway 2高，最终产物为反式5 '-羟基-吡虫啉（trans-5’-hydroxyl-IMI）。第一步质子转移过程是整个代谢反应的速控步骤，计算得到的速控步骤的活化自由能与实验结论一致。以上结果表明，肝酶CYP3A4是IMI生物转化的主要代谢酶。IMI农药在肝酶CYP3A4催化下容易发生代谢转化，生成水溶性更强的羟化代谢物，毒性降低。该结论提示羟化代谢物的进一步代谢活化机理及代谢物的毒性作用不可忽略。该项目的研究结果可为该类杀虫剂的毒性预警和安全应用提供重要理论依据。