Activity‐Based Protein Profiling in Methicillin‐Resistant Staphylococcus aureus Reveals the 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 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 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 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 that could lead to side‐effects and/or contribute to therapeutic efficacy.

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections

“…In an effort to develop selective activity-based probes (ABPs) that can serve as chemical tools to further characterize FphE, we needed to select an optimal electrophile. Among the various moieties capable of directly forming a covalent bond with the active site nucleophile of serine hydrolases, five classes, namely, β-lactam ( 1 ), fluorophosphonate ( 2 ), triazole urea ( 3 ), oxadiazolone ( 4 ), and 1-carbamoyl-5-fluorouracil ( 5 ), have been successfully used in irreversible inhibitors or probes that label FphE (Figure B). ,,− All of these covalent electrophilic warheads are quite distinct, in terms of their mechanisms of action and overall structures. Each has benefits and drawbacks, and some (i.e., triazole ureas, fluorophosphonates, and 5-fluorouracils) have a functional group that is lost upon covalent binding while others (β-lactams and oxadiazolones) do not.…”

Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium

“…The generally low degree of homology with human orthologs is an additional benefit when considering these enzymes as potential antimicrobial drug target candidates for which human off-target activity would not be desired. Given the increasing toolset of reactive electrophile scaffolds for tailored covalent inhibitors targeting specific SHs (Böttcher and Seiber 2008 , Staub and Seiber 2008 , Adibekian et al 2011 , Zuhl et al 2012 , Camara et al 2015 , Lentz et al 2016 , Chen et al 2019 , Babin et al 2022 , Bakker et al 2023 , Uddin et al 2023 ) potentially combined with incorporation of peptidic specificity elements generated by experimental evolution during phage display (Chen et al 2021 ) will make it possible to design specific inhibitors and probes for this underexplored class of E. faecium targets. Such inhibitors may be used as tool compounds in conjunction with genetic tools for functional studies aimed at elucidating the physiological function of these enzymes and may also represent lead compounds for the development of urgently needed treatment options for VRE infections.…”

“…Prior to sample preparation for mass spectrometry samples were frozen at −80°C. The continued sample preparation was performed according to the same procedure as described in Uddin et al ( 2023 ).…”