# Targeting the Cell Wall Salvage Pathway: Dual-Enzyme Inhibition of AmgK and MurU as a Strategy Against Antibiotic Resistance

## TL;DR

This study explores a new strategy to combat antibiotic resistance by targeting two enzymes in Pseudomonas aeruginosa that help it bypass traditional antibiotic treatments.

## Contribution

The paper introduces a dual-enzyme inhibition strategy targeting AmgK and MurU to enhance fosfomycin therapy against drug-resistant bacteria.

## Key findings

- Congo red and CTAB were identified as potent inhibitors of PaMurU through high-throughput screening.
- Molecular docking and SUPR-DSF confirmed that Congo red interacts selectively with PaMurU.
- Ongoing crystallographic studies aim to clarify the atomic-level binding mechanism of Congo red to PaMurU.

## Abstract

The rise of multidrug-resistant Pseudomonas aeruginosa underscores the need for novel therapeutic targets beyond conventional peptidoglycan biosynthesis. Some bacterial strains bypass MurA inhibition by fosfomycin via a cell wall salvage pathway. This study targeted P. aeruginosa AmgK (PaAmgK) and MurU (PaMurU) to identify inhibitors that could complement fosfomycin therapy. A malachite-green-based dual-enzyme assay enabled efficient activity measurements and high-throughput chemical screening. Screening 232 compounds identified Congo red and CTAB as potent PaMurU inhibitors. A targeted mass spectrometric analysis confirmed the selective inhibition of PaMurU relative to that of PaAmgK. Molecular docking simulations indicate that Congo red preferentially interacts with PaMurU through electrostatic contacts, primarily involving the residues Arg28 and Arg202. The binding of Congo red to PaMurU was corroborated further using SUPR-differential scanning fluorimetry (SUPR-DSF), which revealed ligand-induced thermal destabilization. Ongoing X-ray crystallographic studies, in conjunction with site-directed mutagenesis and enzyme kinetic analyses, aim to elucidate the binding mode at an atomic resolution.