Screening Privileged Alkyl Guanidinium Motifs under Host-Mimicking Conditions Reveals a Novel Antibiotic with an Unconventional Mode of Action

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 that do not resemble the infection conditions. To address these limitations, we present a screen of a focused library of alkyl guanidinium 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 identified with activities against Gram-positive and Gram-negative bacteria, highlighting the fidelity of the general concept. We selected one compound (L15) for in-depth mode of action studies that exhibited bactericidal activity against methicillin-resistant Staphylococcus aureus USA300 with a minimum inhibitory concentration of 1.5 μM. Structure-activity relationship studies confirmed the necessity of the guanidinium motif for antibiotic activity. The mode of action was investigated using affinity-based protein profiling with an L15 probe and identified the signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification, docking, and molecular dynamics simulations demonstrated SpsB activation by L15, a recently described mechanism leading to the dysregulation of protein secretion and cell death. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.