The structure-interaction model of polymyxin lipopeptides with human oligopeptide transporter 2
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eLife Assessment
This study presents a potentially important structure-interaction model describing recognition of polymyxin antibiotics by the human transporter hPepT2 and applies these insights to guide the rational design of potent polymyxin B analogues with reduced nephrotoxicity. The conclusions are supported by compelling evidence, integrating molecular dynamics simulations, transporter mutagenesis, uptake assays, protein expression analyses, antibacterial testing, and mouse nephrotoxicity studies, although some mechanistic interpretations remain limited by altered transporter expression and the absence of direct structural validation.
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Abstract
Background
Multidrug-resistant (MDR) Gram-negative bacteria have triggered a critical global health crisis. Polymyxin lipopeptide antibiotics are used as a last-line therapy against these problematic pathogens, but their clinical use is largely limited by severe nephrotoxicity. Human oligopeptide transporter 2 (hPepT2) is a membrane transporter mediating the reabsorption of polymyxins in renal proximal tubular cells, substantially contributing to their nephrotoxicity. However, it remains unclear how polymyxins interact with hPepT2.
Methods
In this study, we investigated the structure-interaction relationship (SIR) of polymyxins with hPepT2 by integrating computational, chemical and cell biology approaches. Bioinformatic modelling predicted the residues essential for the binding of polymyxins with hPepT2. Transporter mutagenesis and molecular analysis were employed to explore the role of each residue in the interaction of hPepT2 and polymyxins. Moreover, we synthesised a series of polymyxin-like analogues with altering the moieties that are critical for binding with hPepT2. The antibacterial activity and nephrotoxicity of these analogues were subsequently assessed.
Results
Our bioinformatic modelling proposed an outward-facing structure of hPepT2 with a possible transport pathway that polymyxins bind to the lateral opening site of hPepT2 (e.g. E214, D215, D317, D342, E622). Molecular assays for transporter function and expression confirmed that D215 residue of hPepT2 is critical for polymyxin binding, while several other residues significantly impact on transporter turnover rate and/or protein expression. Our experimental validations showed that the lipopeptide analogues with altering the Dab1, Dab3, Dab5 and Dab9 moieties of polymyxins demonstrated decreased interactions with hPepT2. Among these synthetic analogues, alanine substitution at Dab3 showed reduced nephrotoxicity in mice while reserved antibacterial activity against a range of bacterial strains.
Conclusions
Overall, this proof-of-concept study demonstrated that the computationally predicted and experimentally validated polymyxin-hPepT2 SIR model provides a viable approach for the discovery of novel, safer lipopeptide antibiotics.
Article activity feed
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eLife Assessment
This study presents a potentially important structure-interaction model describing recognition of polymyxin antibiotics by the human transporter hPepT2 and applies these insights to guide the rational design of potent polymyxin B analogues with reduced nephrotoxicity. The conclusions are supported by compelling evidence, integrating molecular dynamics simulations, transporter mutagenesis, uptake assays, protein expression analyses, antibacterial testing, and mouse nephrotoxicity studies, although some mechanistic interpretations remain limited by altered transporter expression and the absence of direct structural validation.
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Reviewer #1 (Public review):
Summary:
Polymyxins are the last line of drugs to treat gram-negative bacteria-induced multi-drug resistance; however, they cause nephrotoxicity in 60% of patients. In this work, the authors have studied the structure-interaction relationship (SIR) of polymyxins with hPepT2 using computational and experimental methods. Moreover, it is observed that the electrostatic interactions coordinate the hPepT2-Polymyxin interactions; hence, an alanine scanning strategy is used to understand the interactions and derive the polymyxin variants.
Computational methods such as molecular modeling, coarse-grained and all-atom MD simulations, and interaction studies are performed, while the results are validated in the mouse model, which is a great strategy to prove the hypothesis.
Strengths:
A clear understanding of the …
Reviewer #1 (Public review):
Summary:
Polymyxins are the last line of drugs to treat gram-negative bacteria-induced multi-drug resistance; however, they cause nephrotoxicity in 60% of patients. In this work, the authors have studied the structure-interaction relationship (SIR) of polymyxins with hPepT2 using computational and experimental methods. Moreover, it is observed that the electrostatic interactions coordinate the hPepT2-Polymyxin interactions; hence, an alanine scanning strategy is used to understand the interactions and derive the polymyxin variants.
Computational methods such as molecular modeling, coarse-grained and all-atom MD simulations, and interaction studies are performed, while the results are validated in the mouse model, which is a great strategy to prove the hypothesis.
Strengths:
A clear understanding of the hPepT2-Polymyxin interactions and the role of electrostatic interactions is one of the very important strengths of the paper. In addition, this work proposes a great pipeline for using computational approaches and experimental validation methods to guide the development of newer antibiotics.
Overall, the study proposes novel polymyxin analogues with reduced or no nephrotoxicity, thereby providing a promising foundation for the rational development of safer lipopeptide antibiotics.
Weaknesses:
This work is very well executed and presented; however, addressing the following concerns might improve the presentation of the work:
(1) The introduction is well articulated; however, including a paragraph on the known inhibitors might be helpful in understanding the current status. In addition, it might also help to introduce Dabs, FADDI variants, Gly-sar and MIPS.
(2) The following details of modeling with AlphaFold2 should be included: how the final structure was selected, what the RMSD and structure alignment of the template are, and the final selected structure. A section on modeling with all the parameter details might be useful for reproducing the structure. In addition, specify how the alanine scanning was performed alongside the structure prediction of polymyxins.
(3) In the all-atom MD simulation method, detailing several parameters might help in reproducing the results: simulation time for each system, water model, system composition, protonation state, box type and dimensions, salt ions and concentration, membrane parameters and ligand parameterization methods. Also, the following details on energy minimization might be useful: minimization algorithm, number of steps for minimization and structure restraints in place.
(4) On page 6, line 210, the MIC is used for the first time; although MIC is given in the abbreviation list, the first occurrence should have a complete name. A one-line explanation of MIC in the introduction or wherever suitable might be better but is not mandatory.
(5) Similarly, Gly-sar is first mentioned on page 8, line 301, but its complete name is only mentioned later on page 10, line 368. This can be addressed if a short description is included in the introduction section.
(6) For coarse-grained MD simulation, why were 2 replicates performed? Most studies perform 3 replicates, which are also good in terms of statistics and error bar calculations. In addition, the authors should specify whether an independent minimization is done for each of the two replicates or whether the minimization step is common for both.
(7) For MD simulation results, giving simulation movies in supplementary results might be a better way to show how the trajectories behaved.
(8) The description of visualisation software such as VMD or PyMol is missing. The authors should specify if any visualization tool is used.
(9) For the mouse model study, the authors claim that FADDI-795 has no observable nephrotoxicity; however, the n=3 shows that a very small number of mouse models were used to make the assumption. In addition, the number of mice used in each experiment is not explicitly mentioned in the methods section.
(10) In Table 2, the column 8 header is not visible.
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Reviewer #2 (Public review):
Summary:
Jiang et al. sought to elucidate the molecular basis of polymyxin antibiotic interaction with the renal transporter hPepT2, a transporter previously implicated in polymyxin-induced nephrotoxicity. They combined molecular dynamics simulations with transporter mutagenesis, functional uptake assays, kinetic analyses, protein expression studies, antibacterial susceptibility testing, and mouse nephrotoxicity experiments to develop a structure-interaction relationship (SIR) model and apply this model to the rational design of polymyxin analogues.
Overall, the study represents a substantial multidisciplinary effort that integrates computational and experimental approaches. The identification of transporter residues involved in polymyxin recognition and the subsequent design of analogues with reduced …
Reviewer #2 (Public review):
Summary:
Jiang et al. sought to elucidate the molecular basis of polymyxin antibiotic interaction with the renal transporter hPepT2, a transporter previously implicated in polymyxin-induced nephrotoxicity. They combined molecular dynamics simulations with transporter mutagenesis, functional uptake assays, kinetic analyses, protein expression studies, antibacterial susceptibility testing, and mouse nephrotoxicity experiments to develop a structure-interaction relationship (SIR) model and apply this model to the rational design of polymyxin analogues.
Overall, the study represents a substantial multidisciplinary effort that integrates computational and experimental approaches. The identification of transporter residues involved in polymyxin recognition and the subsequent design of analogues with reduced hPepT2-mediated uptake provide a valuable framework for developing safer polymyxin antibiotics. In particular, the identification of FADDI-795 as an analogue that retains antibacterial activity while exhibiting reduced nephrotoxicity represents an encouraging proof of concept.
Strengths:
The computational predictions are strengthened by extensive experimental validation, including site-directed mutagenesis, transport kinetics, fluorescence uptake assays, membrane expression analyses, and in vivo toxicity studies. The consistency between multiple independent experimental approaches increases confidence in many of the authors' conclusions.
Weaknesses:
Several conclusions would benefit from a more cautious interpretation. A major limitation is that several transporter mutations substantially altered total or membrane protein expression, making it difficult to distinguish effects on substrate binding from indirect effects caused by impaired transporter stability or trafficking. The authors acknowledge this limitation in the Discussion, but some mechanistic conclusions remain stronger than the available evidence supports.
Similarly, while the proposed binding model is biologically plausible and supported by mutagenesis, it remains an inferred model derived from molecular simulations rather than a direct structural determination. Statements describing the model as "validated" should therefore be moderated to indicate that the experimental data provide support rather than definitive structural confirmation.
The translational implications are promising but remain preliminary. Although FADDI-795 demonstrated reduced nephrotoxicity in the mouse model while maintaining antibacterial activity, no pharmacokinetic studies were presented to demonstrate reduced renal accumulation or altered tissue distribution, and additional efficacy studies in infection models would further strengthen the therapeutic claims.
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Reviewer #3 (Public review):
Summary:
Jiang et al. described findings aimed at interrogating the interactions of the antibiotic polymyxin B with human kidney proteins that mediate nephrotoxicity. Their findings using both computational molecular dynamics simulations and experimental approaches illustrate the importance of aspartic acid residues (D215) in mediating the antibiotic uptake into the cells, and upon mutagenesis with Alanine, the effects are less pronounced. Further, they could modify the antibiotic units interacting with proteins into less toxic peptides with retained antibacterial properties.
Strengths:
I was impressed by this text, which advances the knowledge of how the antibiotic causes human nephrotoxicity and how this could be exploited into less problematic antibiotic peptides.
Weaknesses:
Interactions of Polymyxin B …
Reviewer #3 (Public review):
Summary:
Jiang et al. described findings aimed at interrogating the interactions of the antibiotic polymyxin B with human kidney proteins that mediate nephrotoxicity. Their findings using both computational molecular dynamics simulations and experimental approaches illustrate the importance of aspartic acid residues (D215) in mediating the antibiotic uptake into the cells, and upon mutagenesis with Alanine, the effects are less pronounced. Further, they could modify the antibiotic units interacting with proteins into less toxic peptides with retained antibacterial properties.
Strengths:
I was impressed by this text, which advances the knowledge of how the antibiotic causes human nephrotoxicity and how this could be exploited into less problematic antibiotic peptides.
Weaknesses:
Interactions of Polymyxin B with kidney proteins were not demonstrable in vivo, and with reliable technologies such as X-ray or NMR.
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