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Proteome-wide profiling of the selectivity of diverse electrophiles (part 2)

ABSTRACT: Targeted covalent inhibitors (TCIs) are established as powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine. Competitive, residue-specific proteomics has been instrumental to streamline covalent drug discovery targeting cysteine. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome-wide comparison of the amino acid selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome-wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor nine different amino acids as well as the N-terminus proteome-wide. This selection e.g. includes the first probes to globally monitor tryptophan, histidine and arginine as well as novel tailored probes for methionine, aspartate and glutamate.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Staphylococcus Aureus

SUBMITTER: Stephan Hacker

LAB HEAD: Dr Stephan M. Hacker

PROVIDER: PXD065811 | Pride | 2025-08-04

REPOSITORIES: Pride

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			Action	DRS
	210507_SMH_210507_P01.raw	Raw
	210507_SMH_210507_P02.raw	Raw
	210507_SMH_210507_P03.raw	Raw
	210507_SMH_210507_P04.raw	Raw
	210507_SMH_210507_P05.raw	Raw

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Project description:Unprecedented bacterial targets are urgently needed for the development of novel antibiotics to overcome the current resistance crisis. Challenges include the limited uptake of compounds as well as prioritizing proteins for their druggability and functional relevance. Especially, the wealth of uncharacterized proteins represents an untapped source for novel targets. However, tools to decipher their function are largely lacking. We here utilize the systematic mining of pyridoxal phosphate dependent enzymes (PLP DEs) in bacteria to focus on a target class, which is known to bind ligands, accesses PLP via active transport from the media and is involved in crucial metabolic processes. For this, we systematically exploited the chemical space of the pyridoxal (PL) scaffold and obtained eight PL probes bearing modifications at various ring positions. These probes were subsequently tested for phosphorylation by cognate kinases and labelling of PLP DEs in clinically relevant Gram-positive (Staphylococcus aureus) as well as Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Overall, the coverage of this diverse set of probes along with refined labelling conditions not only exceeded the performance of a previous probe generation, it also provided a detailed map of binding preferences of certain structural motifs. Although originally conducted in mutant cells devoid of PLP de novo biosynthesis, we here demonstrate efficient PLP DE labelling also in a wild type strain. Overall, the profiling revealed several putative PLP DEs with unknown function, of which we exemplarily characterized five via in-depth enzymatic assays. Finally, we screened a panel of putative PLP binders for antibiotic activity and unravelled the targets of hit molecules via competitive profiling with our probes. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

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Proteome-wide profiling of the selectivity of diverse electrophiles (part 2)

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