Repurposing Azeliragon as a novel antibacterial agent against methicillin-resistant Staphylococcus aureus via combined membrane phospholipids and cell wall targeting.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a persistent clinical threat due to limited therapeutic options and the rapid emergence of resistance. Azeliragon, an orally bioavailable Receptor for Advanced Glycation End-products (RAGE) inhibitor previously tested in human trials, was identified as a potential antibacterial agent against MRSA through high-throughput screening. It exhibited potent antibacterial activity against clinical S. aureus isolates (MIC50 = 6.25 μM/3.3 μg/mL) and significantly inhibited biofilm formation. Metabolomics and proteomics suggested perturbation of lipid pathways and potential interaction with MurA. Target validation using Drug Affinity Responsive Target Stability (DARTS), Determination of biomolecular affinity by bio-layer interferometry (BLI), Limited proteolysisemass spectrometry (LiP-MS), and site-directed mutagenesis provided biochemical evidence supporting MurA as an intracellular target. Whole Genome Sequencing of resistant derivatives revealed Single Nucleotide Polymorphisms (SNPs) in cell-wall and fatty-acid efflux regulators including yycH, farE/farR, consistent with membrane and cell-wall perturbation. In a murine wound model Azeliragon reduced local bacterial burden and accelerated healing to levels comparable with vancomycin. Azeliragon is a repurposable antibacterial agent that perturbs membrane phospholipid homeostasis and engages MurA-dependent peptidoglycan synthesis. These findings support further preclinical development of Azeliragon or optimized derivatives as potential therapeutics against MRSA and other gram-positive pathogens.