Department of Medical Biology, UiT- The Arctic University of Norway, 9019, Tromsø, Norway.
Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Chembiochem. 2023 Nov 2;24(21):e202300473. doi: 10.1002/cbic.202300473. Epub 2023 Aug 29.
Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy.
基于活性的蛋白质谱分析是一种强大的化学蛋白质组学技术，可用于检测活性酶，并鉴定药物的靶标和非靶标。在这里，我们报告了使用卡莫氟和基于活性的探针来鉴定细菌病原体金黄色葡萄球菌中具有生物学相关性的酶。卡莫氟是 5-氟尿嘧啶的抗肿瘤前药，也具有抗菌和抗生物膜活性。卡莫氟探针最初是为靶向人类酸性神经酰胺酶而设计的，该酶是 NTN 水解酶家族的成员，具有活性位点半胱氨酸亲核试剂。在这里，我们首先在生物膜促进条件下和液体培养中对活的金黄色葡萄球菌中的荧光卡莫氟探针的靶标进行了分析，然后通过液相色谱/质谱进行靶标鉴定。用卡莫氟-生物素探针处理导致 20 种来自不同家族的酶的富集，这些酶有待进一步表征，包括与 NTN 水解酶相关的 IMP 环水解酶 PurH。然而，该探针优先标记丝氨酸水解酶，因此显示出与氨基甲酸酯相似的反应性谱。我们的结果表明，亲电的 N-碳酰胺-5-氟尿嘧啶支架有可能被优化，以实现对不同酶家族的选择性。观察到的混杂反应性谱表明，卡莫氟的临床应用可能导致许多人和微生物酶的失活，这可能导致副作用和/或有助于治疗效果。
