Bioinformatically-derived NRP A8NO5 destroys *Staphylococcus epidermidis* cell membranes, showing potent antibacterial activity.

The escalating global crisis of antibiotic resistance, particularly in hospital-acquired infections caused by bacteria like Staphylococcus epidermidis, necessitates the urgent discovery of novel antimicrobial agents. Current antibiotic treatments are increasingly ineffective, leading to prolonged hospital stays and higher mortality rates. Antimicrobial Peptides (AMPs) offer a promising alternative due to their distinct mechanisms of action, often targeting bacterial cell membranes, which can circumvent existing resistance pathways. However, traditional AMP discovery through bioassay-guided fractionation is often hampered by the difficulty of culturing many microorganisms and the sheer complexity of natural product isolation.

Researchers mined thousands of non-ribosomal peptide synthetase (NRPS) biosynthetic gene clusters (BGCs) from hundreds of Rhodococcus genomes in public databases. Bioinformatic tools were then used to predict the core molecular skeletons of non-ribosomal peptides (NRPs) synthesized by selected NRPSs. An initial NRP analogue, A8-5-line, was obtained via chemical synthesis and tested for activity against S. epidermidis. Subsequent structure-activity relationship (SAR) studies guided modifications, specifically substituting the C-terminal hydrophilic glutamine with a hydrophobic alanine, to generate the derivative A8NO5. Mechanistic studies investigated bacterial cell membrane integrity and performed metabolomic analysis. In vitro cytotoxicity and hemolytic activity assays were also conducted to assess biosafety.

The initial chemically synthesized NRP analogue, A8-5-line, demonstrated potent activity against Staphylococcus epidermidis. Subsequent structure-activity relationship (SAR) studies proved crucial, as the modification of A8-5-line to A8NO5 by substituting a C-terminal hydrophilic glutamine with a hydrophobic alanine significantly enhanced its antibacterial activity. Mechanistic investigations revealed that A8NO5 effectively destroys bacterial cell membrane integrity, a critical mode of action for many potent AMPs. This membrane disruption was further supported by metabolomic analysis, which showed that the nucleotide metabolism pathway was disrupted upon A8NO5 treatment. > The altered nucleotide-related metabolites are likely downstream effects of the primary membrane damage and the associated metabolic collapse within the bacterial cell. Importantly, A8NO5 displayed negligible cytotoxicity and hemolytic activity in vitro, indicating a favorable biosafety profile for a potential therapeutic agent.