Expanding the therapeutic window of gramicidin S towards a safe and effective systemic treatment of methicillin-resistant S. aureus infections.

The rise of multidrug-resistant bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), necessitates the development of new antibacterial therapies. Antimicrobial peptides offer a promising alternative to conventional antibiotics due to their unique mechanisms of action. Gramicidin S exhibits potent bactericidal activity against S. aureus, however, high haemolytic toxicity currently limits its application to topical use. A new series of gramicidin S analogues is presented with rational modifications to the β-turn and β-strand regions, to reduce haemolytic and nephrotoxic effects, while preserving antibacterial potency. The minimum inhibitory concentration (MIC) for each analogue was determined against benchmark methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, with toxicity characterised in vitro using human red blood cells and human embryonic kidney cells (HEK-293). Peptide 12 demonstrated a significant two-fold increase in antibacterial activity against both MSSA and MRSA (MIC: 2 μg/mL) compared to gramicidin S (MIC: 4 μg/mL), albeit with increased cytotoxicity. Similarly, peptide 15 showed exceptional efficacy (MIC: 3 μg/mL), but with reduced cytotoxicity, culminating in a two-fold improvement to the therapeutic index (TI) of gramicidin S. Peptides 14 (HC: 50.48 ± 1.15 μg/mL, IC: 38.09 μg/mL) and 16 (HC: 84.09 ± 1.02 μg/mL, IC: 12.60 μg/mL) also significantly reduced haemolytic toxicity and nephrotoxicity, compared to gramicidin S (HC: 12.34 ± 0.27 μg/mL, IC: 6.45 μg/mL). Detailed NMR, CD and computational modelling were used to provide critical insights into how molecular conformation influences both antibacterial potency and cytotoxicity. Collectively, these results expand the therapeutic window of gramicidin S by up to 12-fold, with negligible cytotoxicity observed at concentrations well beyond the acceptable safety threshold, which indicates the potential for safe systemic administration in the treatment of infection caused by resistant pathogens.