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Screening privileged alkylguanidinium motifs under host-mimicking conditions reveals a novel antibiotic with an unconventional mode of action

ABSTRACT: Screening large molecule libraries against pathogenic bacteria is often challenged by a low hit rate due to limited uptake, underrepresentation of antibiotic structural motifs and assays which do not resemble the infection conditions. To address these limitations, we here screen a library focused on alkylguanidinium compounds, a structural motif associated with antibiotic activity and enhanced uptake, under host-mimicking infection conditions against a panel of disease-associated bacteria. Several hit molecules were obtained with activities against Gram-positive as well as Gram-negative bacteria highlighting the fidelity of the general concept. Based on the best antibiotic activity, we selected one compound (L15) for in-depth mode of action studies. L15 exhibited bactericidal activity against Staphylococcus aureus USA300 Lac (JE2) with a minimum inhibitory concentration of 1.5 µM whereby a structure-activity relationship study with 18 derivatives proved the necessity of the guanidinium motif for antibiotic activity. Electron microscopy studies and depolarization assays at high L15 concentrations showed an impact on the cell membrane and integrity which was absent at the MIC concentration. Sequencing of L15-resistant strains revealed a mutation in an efflux pump but did not provide hints for direct targets. We thus performed affinity-based protein profiling with a L15 probe and identified signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification and molecular docking demonstrated SpsB overactivation by L15, similar to a previously reported activator molecule that dysregulates protein secretion of this essential enzyme. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.

INSTRUMENT(S): Orbitrap Eclipse, timsTOF Pro

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Nina Bach

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD051986 | Pride | 2024-07-17

REPOSITORIES: Pride

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			Action	DRS
	12815_9-25-2023_DS_USA300_FP13_5_5h_1_5_1_GA1.d.zip	Other
	12817_9-25-2023_DS_USA300_FP13_5_5h_1_5_2_GA2.d.zip	Other
	12819_9-25-2023_DS_USA300_FP13_5_5h_1_5_3_GA3.d.zip	Other
	12821_9-25-2023_DS_USA300_FP13_5_5h_1_5_4_GA4.d.zip	Other
	12823_9-25-2023_DS_USA300_FP13_5_5h_3_1_GA5.d.zip	Other

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Screening privileged alkylguanidinium motifs under host-mimicking conditions reveals a novel antibiotic with an unconventional mode of action

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