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

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 on the use of a carmofur-based activity-based probes to identify biologically relevant enzymes in the bacterial pathogenStaphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also hasantimicrobial 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 liveS. aureusunder biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatograpy/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 labelled serine hydrolases, thus displaying a reactivity profile similar to carbamates. Our results suggest that the electrophilicN-carbamoyl-5-fluorouracilscaffold 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 that could lead to side-effects and/or contribute to therapeutic efficacy.