Activity‐Based Protein Profiling in Methicillin‐Resistant Staphylococcus aureus Reveals the Broad Reactivity of a Carmofur‐Derived Probe

ChemBioChem, vol. 24, pp. e202300473

Abstract

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.

Authors 3

1. Md Jalal Uddin

   UiT The Arctic University of Norway

   Affiliation as printed

   Department of Medical Biology UiT- The Arctic University of Norway 9019 Tromsø Norway

2. Herman S. Overkleeft Aachen

   Leiden University

   Affiliation as printed

   Department of Bioorganic Synthesis Leiden Institute of Chemistry Leiden University Einsteinweg 55 2333 CC Leiden The Netherlands

   Department of Bioorganic Synthesis Leiden Institute of Chemistry Leiden University Einsteinweg 55 2333 CC Leiden The Netherlands

3. Christian Stephan Lentz corresponding

   UiT The Arctic University of Norway

   Affiliation as printed

   Department of Medical Biology UiT- The Arctic University of Norway 9019 Tromsø Norway