MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology , University of Liverpool , Liverpool L69 3GE , U.K.
GlaxoSmithKline , Park Road , Ware , Hertfordshire SG12 0DP , U.K.
Chem Res Toxicol. 2019 Oct 21;32(10):2095-2106. doi: 10.1021/acs.chemrestox.9b00282. Epub 2019 Sep 11.
Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.
磺胺甲恶唑（SMX）治疗时常引起过敏反应。这些不良反应归因于亚硝基磺胺甲恶唑（SMX-NO），它是代谢物磺胺甲恶唑羟胺自氧化形成的反应产物。SMX-NO 在体外刺激幼稚 T 细胞以及激活来自过敏患者的 T 细胞的能力表明，T 细胞的激活可能通过 SMX-NO 与蛋白质结合或通过直接修饰 MHC 结合的肽来发生。SMX-NO 已被证明可以在体外修饰谷胱甘肽、设计肽和蛋白质中的半胱氨酸残基；然而，这些加合物在体内尚未被表征。在这项研究中，使用质谱法对 SMX-NO 加合物进行了平行的体外和体内分析。除了已知的半胱氨酸加合物外，在体外还发现了多个 SMX-NO 衍生的半抗原结构存在于人血清白蛋白（HSA）的赖氨酸和酪氨酸残基上。在赖氨酸残基上，鉴定出两种半抗原结构，包括芳基偶氮烷加合物和席夫碱加合物。有趣的是，这些加合物对热不稳定且易水解，如 allysine 的存在所示。此外，在长期接受 SMX 治疗的患者中检测到 SMX 修饰的 HSA 加合物，这表明存在源自 SMX-NO 的拟羧酸代谢物的芳基偶氮烷加合物。这些加合物的存在可以解释 SMX 的免疫原性以及在 T 细胞培养测定中观察到的对 SMX-NO 的强烈反应。此外，这些加合物降解为 allysine 可能导致与 T 细胞激活所需的应激相关的先天免疫反应。
