Dual-Target Compound 4E2RCat Combats MRSA by Disrupting Membrane Homeostasis and Targeting the Virulence Regulator ClpX

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) poses a severe global health threat due to multidrug resistance, biofilm formation, and potent virulence. The ATP-dependent Clp protease subunit ClpX plays a central role in proteostasis and virulence regulation, making it an attractive target for anti-MRSA therapy. Here, we identified 4E2RCat, a previously reported inhibitor of eukaryotic translation initiation factor 4E (eIF4E), as a dual-functional antimicrobial agent. It exerted antibacterial activities by disrupting bacterial membrane homeostasis and attenuated virulence by directly targeting of ClpX. 4E2RCat displayed robust antibacterial activity against MRSA, with minimal inhibitory concentrations (MICs) ranging from ≤ 3.13 to 12.5 μM, and conferred a low propensity to resistance development. Moreover, it inhibited biofilm formation, eradicated mature biofilms, and killed intracellular MRSA and persister cells. Mechanistically, transcriptomic and membrane assays revealed that 4E2RCat perturbed bacterial membrane lipid homeostasis, reduced membrane fluidity, increased permeability, and induced oxidative stress. Drug affinity responsive target stability, biolayer interferometry, molecular docking, and gene knockout assays identified ClpX as one of the direct cellular targets of 4E2RCat, through which the compound suppressed the expression of key virulence factors, including AgrA, α-hemolysin, and SaeR. In a murine model of MRSA pneumonia, 4E2RCat markedly improved mouse survival, reduced pulmonary bacterial burden, and alleviated lung tissue damage. These findings establish 4E2RCat as a multitargeting lead compound whose combination of rapid membrane homeostasis disruption and ClpX-mediated virulence attenuation underlies its low propensity for resistance development, supporting the development of dual-targeting therapeutics against drug-resistant MRSA infections.