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. 2019 Sep 20;14(9):2065-2070.
doi: 10.1021/acschembio.9b00565. Epub 2019 Sep 12.

Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis

Alexandra Antonoplis  1 , Xiaoyu Zang  1 , Tristan Wegner  2 , Paul A Wender  1   3 , Lynette Cegelski  1
Affiliations

Vancomycin-Arginine Conjugate Inhibits Growth of Carbapenem-Resistant E. coli and Targets Cell-Wall Synthesis

Alexandra Antonoplis et al. ACS Chem Biol. .

Abstract

The emergence of multi-drug-resistant Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae, is a major health problem that necessitates the development of new antibiotics. Vancomycin inhibits cell-wall synthesis in Gram-positive bacteria but is generally ineffective against Gram-negative bacteria and is unable to penetrate the outer membrane barrier. In an effort to determine whether vancomycin and other antibiotics effective against Gram-positive bacteria could, through modification, be rendered effective against Gram-negative bacteria, we discovered that the covalent attachment of a single arginine to vancomycin yielded conjugates with order-of-magnitude improvements in activity against Gram-negative bacteria, including pathogenic E. coli. The vancomycin-arginine conjugate (V-R) exhibited efficacy against actively growing bacteria, induced the loss of rod cellular morphology, and resulted in the intracellular accumulation of peptidoglycan precursors, all consistent with cell-wall synthesis disruption as its mechanism of action. Membrane permeabilization studies demonstrated an enhanced outer membrane permeability of V-R as compared with vancomycin. The conjugate exhibited no mammalian cell toxicity or hemolytic activity in MTT and hemolysis assays. Our study introduces a new vancomycin derivative effective against Gram-negative bacteria and underscores the broader potential of generating new antibiotics through combined mode-of-action and synthesis-informed design studies.

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Figures

Figure 1.
Figure 1.
Preparation and antibacterial activity of vancomycin-amino acid conjugates. R corresponds to each amino acid amide connected to vancomycin (left). An MIC activity screen was performed in E. coli 25922 (right), where V-O, V-K, and V-D exhibited MICs of 32 μM or higher and were not further examined in subsequent experiments. Data for V-R, V-K, V-RR, and vancomycin represents median MICs obtained from 2–3 independent experiments, with ranges provided in parentheses.
Figure 2.
Figure 2.
V-R targets actively growing E. coli and inhibits cell-wall synthesis. A) Time-kill kinetic analysis of V and V-R-treated UTI89 in Mueller-Hinton broth. Treatment concentrations were 16 μM for V-R and 256 μM for V. B) Both V and V-R treatment result in alteration of E. coli 25922 morphology, inducing rounding of bacterial cells (scale bar: 10 μm). Treatment concentrations were 16 μM for V-R and 256 μM for V. C & D) V-R treatment results in accumulation of UDP-MurNAc peptide in UTI89 as identified by HPLC and mass spectrometry. In C, treatment concentrations were 16 μM for V-R and 16 μM for V.
Figure 3.
Figure 3.
Impacts of V-R on outer and inner membrane integrity in E. coli. A) Outer membrane permeabilization assay in UTI89 using the fluorescent probe, NPN. B) Outer and inner membrane permeabilization assay in UTI89, where propidium iodide was used as probe of membrane perturbation. In A and B, treatment concentrations were performed at 4X MIC (4 μM for polymyxin B, 512 μM for V and 64 μM for V-R).
See this image and copyright information in PMC

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