# Self-Stabilizing Covalent Ligand Targets Bacterial Phosphatidylethanolamine and Enhances Antibiotic Efficacy

## TL;DR

A new covalent ligand called DMAX selectively targets bacterial membranes and enhances antibiotic effectiveness without harming mammalian cells.

## Contribution

DMAX is a novel covalent ligand that selectively targets phosphatidylethanolamine in bacterial membranes, enabling enhanced antibiotic efficacy.

## Key findings

- DMAX selectively interacts with phosphatidylethanolamine through covalent and ionic bonds, as confirmed by spectroscopic and computational analyses.
- Fluorescently labeled DMAX binds to PE-enriched membranes and Gram-negative bacteria while avoiding mammalian cells.
- Conjugating DMAX to Gemifloxacin increases antibiotic efficacy tenfold, even after rapid drug clearance.

## Abstract

Background/Objectives: Discriminating bacterial from mammalian membranes remains a central challenge in antibiotic design. Bacterial membranes are enriched in phosphatidylethanolamine (PE), a lipid normally absent from the outer leaflet of mammalian cells, providing a signature for selective molecular engagement. We report a compact covalent ligand, 6-dimethylamino-4-ketohexanoic acid (DMAX), which targets PE via Schiff base formation, leveraging its tertiary amine to facilitate the reaction and strengthen ionic binding with the phosphate group. Methods: The reactivity of DMAX and PE was evaluated by computational simulations, and their interaction was examined by spectroscopic analyses (NMR and FT-IR) and an artificial membrane assay. The targeting ability of DMAX for live bacteria was determined by microscopy study, and its applicability to therapeutic system was tested in vitro under washed conditions that mimic rapid in vivo clearance. Results: Spectrometric analyses revealed the selective covalent interaction of DMAX and PE, consistent with the simulated results. Fluorescently labeled DMAX selectively binds PE-enriched model membranes and efficiently recognizes Gram-negative bacteria while sparing mammalian cells. Conjugation of DMAX to Gemifloxacin (Gem) significantly enhanced antibiotic efficacy by 10-fold compared with free Gem, even after rapid drug clearance, while maintaining safety in mammalian cells. Conclusions: These results identify DMAX as an efficient and versatile PE-targeting platform, enabling selective membrane anchoring to advance precision antibiotic strategies.