Lateral opening site of human oligopeptide transporter 2 plays a key role in the interaction with polymyxins

Multidrug-resistant (MDR) Gram-negative bacteria pose a critical global health threat, while polymyxins remain a last-line therapy. However, their clinical use is limited by nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal cells and contributes to their nephrotoxicity, but the molecular basis of their interaction remains unclear. Here, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical, and cell biology approaches. Bioinformatic modeling predicted an outward-facing hPepT2 structure and a potential transport pathway, with polymyxins interacting at the lateral opening, particularly E214, D215, D317, D342, and E622. Transporter mutagenesis and molecular analyses confirmed that D215 is critical for polymyxin binding, while other residues influence transporter turnover and/or expression. We subsequently synthesized polymyxin analogs with modifications at Dab1, Dab3, Dab5, and Dab9 of polymyxins, which reduced interactions with hPepT2. Notably, alanine substitution at Dab3 reduced nephrotoxicity in mice while retaining antibacterial activity. Overall, this proof-of-concept study demonstrates that the hPepT2-polymyxin SIR model provides a viable strategy for developing novel, safer lipopeptide antibiotics.