Dataset Information

Indolin-2-one nitroimidazole antibiotics exhibit an unexpected dual mode of action

ABSTRACT: Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon in vivo reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound treated cells, revealing an upregulation of bacteriophage-associated proteins, indicative of DNA damage. Since DNA binding of the compound was not observed, we applied activity-based protein profiling (ABPP) for direct target discovery. Labeling studies revealed topoisomerase IV, an essential enzyme for DNA replication, as the most enriched hit in pathogenic Staphylococcus aureus cells. Subsequent topoisomerase assays confirmed the inhibition of DNA decatenation in the presence of indolin-2-one nitroimidazole with an activity comparable to ciprofloxacin, a known inhibitor of this enzyme. Furthermore, we determined significantly increased redox potentials of indolin-2-one nitroimidazoles compared to classic 5-nitroimidazoles such as metronidazole, which facilitates in vivo reduction. Overall, this study unravels that indolin-2-one functionalized nitroimidazoles feature an unexpected dual MoA, which impairs resistance development. Given the clinical application of this compound class, the new mechanism could be a starting point to mitigate resistance.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Nina Bach

LAB HEAD: Stephan A. Sieber

PROVIDER: PXD033128 | Pride | 2022-11-09

REPOSITORIES: Pride

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Publications

Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action.

Reinhardt Till T Lee Kyu M KM Niederegger Lukas L Hess Corinna R CR Sieber Stephan A SA

ACS chemical biology 20221019 11

Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon <i>in vivo</i> reduction of the nitro group, reactive radicals damage DNA and proteins in the absence of oxygen. Unexpectedly, a recent study of nitroimidazoles linked to an indolin-2-one substituent revealed potent activities against aerobic bacteria. This suggests a different, yet undiscovered mode of action (MoA). To decipher this MoA, we first performed whole proteome analysis of compound- ...[more]

PMID: 36259427

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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.

Dataset Information

Indolin-2-one nitroimidazole antibiotics exhibit an unexpected dual mode of action

Publications

Indolin-2-one Nitroimidazole Antibiotics Exhibit an Unexpected Dual Mode of Action.

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