Drug-induced toxicity has, in many cases, been linked to oxidative metabolism resulting in the formation of reactive metabolites and subsequent covalent binding to biomolecules. Two structurally related antipsychotic drugs, clozapine (CLZ) and olanzapine (OLZ), are known to form similar nitrenium ion reactive metabolites. CLZ-derived reactive metabolites have been linked to agranulocytosis and hepatotoxicity. We have studied the oxidative metabolism of CLZ and OLZ as well as two known metabolites of CLZ, desmethyl-CLZ (DCLZ), and CLZ-N-oxide (CLZ-NO), using in vitro rat liver microsomal (RLM) incubations with glutathione (GSH) trapping of reactive metabolites and liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Reactive metabolite binding to selected standard peptides and recombinant purified human proteins was also evaluated. Bottom-up proteomics was performed using two complementary proteases, prefractionation of peptides followed by LC-HRMS/MS for elucidating modifications of target proteins. Induced RLM was selected to form reactive metabolites enzymatically to assess the complex profile of reactive metabolite structures and their binding potential to standard human proteins. Multiple oxidative metabolites and several different GSH adducts were found for CLZ and OLZ. Modification sites were characterized on human glutathione S-transferase (hGST) alpha 1 (OLZ-modified at Cys112), hGST mu 2 (OLZ at Cys115), and hGST pi (CLZ, DCLZ, CLZ-NO and OLZ at Cys170), human microsomal GST 1 (hMGST1, CLZ and OLZ at Cys50), and human serum albumin (hSA, CLZ at Cys34). Furthermore, two modified rat proteins, microsomal GST 1 (CLZ and OLZ at Cys50) and one CYP (OLZ-modified, multiple possible isoforms), from RLM background were also characterized. In addition, direct effects of the reactive metabolite modifications on proteins were observed, including differences in protease cleavage specificity, chromatographic behavior, and charge-state distributions.

中文翻译：

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通过液相色谱-串联质谱法研究氯氮平和奥氮平反应性代谢物的形成和蛋白质结合。

在许多情况下，药物引起的毒性与氧化代谢有关，导致反应性代谢物的形成和随后与生物分子的共价结合。已知两种结构相关的抗精神病药物氯氮平 (CLZ) 和奥氮平 (OLZ) 可形成类似的氮铵离子反应性代谢物。CLZ 衍生的反应性代谢物与粒细胞缺乏症和肝毒性有关。我们研究了 CLZ 和 OLZ 的氧化代谢以及 CLZ 的两种已知代谢物，脱甲基-CLZ (DCLZ) 和CLZ- N-氧化物 (CLZ-NO)，使用体外大鼠肝微粒体 (RLM) 孵化与谷胱甘肽 (GSH) 捕获反应性代谢物和液相色谱-高分辨率串联质谱 (LC-HRMS/MS)。还评估了与选定的标准肽和重组纯化的人类蛋白质结合的反应性代谢物。自下而上的蛋白质组学使用两种互补蛋白酶进行，肽的预分馏，然后是 LC-HRMS/MS，用于阐明目标蛋白质的修饰。选择诱导 RLM 以酶促形成反应性代谢物，以评估反应性代谢物结构的复杂特征及其与标准人类蛋白质的结合潜力。对于 CLZ 和 OLZ，发现了多种氧化代谢物和多种不同的 GSH 加合物。修饰位点在人谷胱甘肽S上表征-转移酶 (hGST) alpha 1（在 Cys112 处 OLZ 修饰）、hGST mu 2（在 Cys115 处的 OLZ）和 hGST pi（在 Cys170 处的 CLZ、DCLZ、CLZ-NO 和 OLZ）、人微粒体 GST 1（hMGST1、CLZ 和Cys50 处的 OLZ）和人血清白蛋白（hSA，Cys34 处的 CLZ）。此外，还表征了来自 RLM 背景的两种修饰的大鼠蛋白质，微粒体 GST 1（Cys50 处的 CLZ 和 OLZ）和一种 CYP（OLZ 修饰的，多种可能的同种型）。此外，还观察到反应性代谢物修饰对蛋白质的直接影响，包括蛋白酶切割特异性、色谱行为和电荷状态分布的差异。