Lateral opening site of human oligopeptide transporter 2 plays a key role in the interaction with polymyxins
Abstract
Background
Multidrug-resistant (MDR) Gram-negative bacteria have presented a critical global health crisis and polymyxins remain a last-line therapy. However, their clinical use has been largely limited by nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal cells, contributing to their nephrotoxicity. However, it remains unclear how polymyxins interact with hPepT2.
Methods
We investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical, and cell biology approaches. Bioinformatic modelling predicted the essential residues of hPepT2 for the binding to polymyxins. Transporter mutagenesis and molecular analysis were employed to examine the role of each residue in the interaction between hPepT2 and polymyxins. Subsequently, we synthesised a series of new analogues with alterations to the moieties of the polymyxin scaffold that are critical for binding with hPepT2, and assessed their antibacterial activity and nephrotoxicity.
Results
Our bioinformatic modelling proposed an outward-facing structure of hPepT2 with a possible transport pathway, in which polymyxins bind to the lateral opening site of hPepT2, particularly E214, D215, D317, D342, and E622. Molecular assays for transporter function and expression confirmed that D215 of hPepT2 is critical for polymyxin binding, while several other residues significantly impact transporter turnover rate and/or protein expression. Our experimental validations showed that the lipopeptides with alterations to the Dab1, Dab3, Dab5, and Dab9 residues of polymyxins demonstrated decreased interactions with hPepT2. Among these analogues, alanine substitution at Dab3 showed reduced nephrotoxicity in mice while retaining antibacterial activity.
Conclusions
Overall, this proof-of-concept study demonstrated that our SIR model of polymyxins with hPepT2 provides a viable approach for the discovery of novel, safer lipopeptide antibiotics.
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