斑蝥素是一种来自鞘翅目甲虫的萜类化合物。斑蝥素已用于治疗传染性软疣和某些类型的肿瘤。斑蝥素具有剧毒，世界各地都有斑蝥素中毒和死亡的病例报道。斑蝥素引起的毒性的机制仍不清楚。斑蝥素含有酸酐，可能与生物胺发生反应。本研究旨在检测斑蝥素对亲核试剂的化学反应性，并在体外和小鼠体内表征斑蝥素与生物胺的加合物。在这里，斑蝥素在生理条件下分别与游离氨基酸、模拟肽或含胺化合物一起孵育，形成两种类型的缀合物。酰胺型缀合物是通过斑蝥素酸酐与生物胺的伯氨基结合产生的。酰亚胺型缀合物由酰胺型缀合物脱水和环化生成。使用高分辨率质谱法对缀合物的结构进行了表征。我们引入了 14N/15N 和 79Br/81Br 同位素特征，以分别使用 L-(ε)15N-赖氨酸、L-赖氨酸-15N2 和溴标记肼确认缀合物的形成。 NMR实验也证实了酰亚胺缀合物的结构。此外，在小鼠肝脏和尿液中检测到斑蝥素与氨基酸或N-乙酰基赖氨酸的酰胺和酰亚胺缀合物。斑蝥素被发现可以修饰小鼠肝脏中的赖氨酸残基蛋白。 Pan-P450 抑制剂 1-氨基苯并三唑显着增加尿液斑蝥素-N-乙酰基-赖氨酸缀合物，同时减少斑蝥素代谢物。总之，斑蝥素酸酐可以非酶促地与生物胺共价结合，这有助于更好地理解非酶促反应性在斑蝥素中毒中的作用。意义说明 斑蝥素的酸酐部分可以非酶促地与生物胺的伯氨基共价结合。斑蝥素酸酐与氨基酸的伯氨基、模拟肽和蛋白质赖氨酸残基共价结合后生成酰胺和酰亚胺缀合物。使用同位素标记试剂和 NMR 实验，通过 14N/15N 和 79Br/81Br 同位素特征确认了缀合物的结构。这项研究将有助于了解非酶反应性在斑蝥素中毒中的作用。版权所有 © 2024 美国药理学和实验治疗学会。

Cantharidin is a terpenoid from coleoptera beetles. Cantharidin has been used to treat molluscum contagiosum and some types of tumors. Cantharidin is highly toxic and cantharidin poisoning and fatal cases have been reported worldwide. The mechanisms underlying cantharidin-induced toxicity remain unclear. Cantharidin contains anhydride, which may react with biological amines. This study aimed to examine the chemical reactivity of cantharidin toward nucleophiles and characterize adducts of cantharidin with biological amines in vitro and in mice. Here, two types of conjugates were formed in the incubation of cantharidin under physiologic conditions with free amino acids, a mimic peptide, or amine-containing compounds, respectively. Amide-type conjugates were produced by the binding of cantharidin anhydride with the primary amino group of biological amines. Imide-type conjugates were generated from the dehydration and cyclization of amide-type conjugates. The structure of the conjugates was characterized by using the high-resolution mass spectrometry. We introduced the 14N/15N and 79Br/81Br isotope signatures to confirm the formation of conjugates using L-(ε)15N-lysine, L-lysine-15N2, and bromine-tagged hydrazine, respectively. The structure of imide conjugate was also confirmed by NMR experiments. Furthermore, the amide and imide conjugates of cantharidin with amino acids or N-acetyl-lysine were detected in mouse liver and urine. Cantharidin was found to modify lysine residue proteins in mouse liver. Pan-P450 inhibitor 1-aminobenzotriazole significantly increased the urine cantharidin-N-acetyl-lysine conjugates whereas decreased cantharidin metabolites. In summary, cantharidin anhydride can covalently bind to biological amines nonenzymatically, which facilitates a better understanding of the role of nonenzymatic reactivity in cantharidin poisoning. Significance Statement Anhydride moiety of cantharidin can covalently bind to the primary amino group of biological amines nonenzymatically. Amide and imide conjugates were generated after the covalent binding of cantharidin anhydride with the primary amino groups of amino acids, a mimic peptide, and protein lysine residues. The structure of conjugates was confirmed by 14N/15N and 79Br/81Br isotope signatures using isotope-tagged reagents and NMR experiments. This study will facilitate the understanding of the role of nonenzymatic reactivity in cantharidin poisoning.Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.