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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 probe to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug with antimicrobial activity that is related to 5-fluorouracil. The carmofur-probe was 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 21 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 or even contribute to therapeutic efficacy.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Bacteria Staphylococcus Aureus

DISEASE(S): Commensal Bacterial Infectious Disease

SUBMITTER: Md Jalal Uddin

LAB HEAD: Christian S. Lentz

PROVIDER: PXD043275 | Pride | 2024-01-29

REPOSITORIES: Pride

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			Action	DRS
	JUddin_SA_carmofur_ABPP.msf	Msf
	JUddin_SA_carmofur_ABP_Biotin_R1.raw	Raw
	JUddin_SA_carmofur_ABP_Biotin_R2.raw	Raw
	JUddin_SA_carmofur_ABP_Biotin_R3.raw	Raw
	JUddin_SA_carmofur_ABP_Cntrl_R1.raw	Raw

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Project description:Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-targets, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy, PhosID-ABPP, on a timsTOF Pro mass spectrometer. This refinement enables dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely PF-06672131 and PF-6422899, facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, while both ABPs showed comparable affinities for the EGFR, PF-06672131 had a broader off-target reactivity profile. In contrast, PF-6422899 exhibited higher affinity for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF-06672131 also effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nanomolar. Additionally, analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

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

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