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

Area of Science:

• Microbiology
• Drug Discovery
• Biochemistry

Background:

• Multidrug-resistant *Pseudomonas aeruginosa* poses a significant clinical challenge.
• Conventional therapies targeting peptidoglycan biosynthesis are becoming less effective due to resistance mechanisms.
• Bacterial strains can evade fosfomycin by utilizing cell wall salvage pathways involving enzymes like MurA.

Purpose of the Study:

• To identify novel inhibitors targeting *Pseudomonas aeruginosa* AmgK (*Pa*AmgK) and MurU (*Pa*MurU).
• To find compounds that can complement fosfomycin therapy and overcome resistance.
• To explore alternative therapeutic strategies against multidrug-resistant *P. aeruginosa*.

Main Methods:

• A malachite-green-based dual-enzyme assay was developed for high-throughput screening.
• 232 compounds were screened to identify inhibitors of *Pa*AmgK and *Pa*MurU.
• Targeted mass spectrometry, molecular docking, and SUPR-differential scanning fluorimetry (SUPR-DSF) were used to validate findings.

Main Results:

• Congo red and CTAB were identified as potent inhibitors of *Pa*MurU.
• Selective inhibition of *Pa*MurU over *Pa*AmgK was confirmed.
• Molecular docking suggested that Congo red binds to *Pa*MurU via electrostatic interactions with key residues (Arg28, Arg202).
• SUPR-DSF confirmed ligand-induced thermal destabilization of *Pa*MurU upon Congo red binding.

Conclusions:

• Congo red and CTAB represent promising lead compounds for targeting *Pa*MurU.
• Inhibiting *Pa*MurU offers a potential strategy to combat fosfomycin resistance in *P. aeruginosa*.
• Further structural and enzymatic studies are underway to fully elucidate the inhibition mechanism.