Repurposing GSK2018682 Confers Dual Antibacterial and Antibiofilm Activity against <i>Staphylococcus aureus</i>

The increase in the level of antibiotic-resistant <i>Staphylococcus aureus</i> underscores the urgent need for novel therapeutics. Consequently, repurposing clinically approved drugs has emerged as a compelling and time-saving strategy to counteract this escalating antimicrobial crisis. Here, high-throughput screening revealed that GSK2018682, an agonist of sphingosine-1-phosphate receptors S1P1 and S1P5, exhibits potent, broad-spectrum antimicrobial activity, particularly against <i>S. aureus</i> (MIC = 25-50 μM) and <i>E. faecalis</i> (MIC = 12.5-25 μM). GSK2018682 exhibited concentration-dependent bactericidal activity against both MSSA and MRSA, eradicating planktonic <i>S. aureus</i> within 6 h at 4× MIC. At sub-MIC concentrations, GSK2018682 significantly inhibited biofilm formation, and at 4× MIC, it killed the bacterial cells embedded in mature biofilms. Proteomics revealed that GSK2018682 caused global expression perturbations of functional proteins involved in a two-component system, various metabolic pathways, ribosomal functions, and biofilm regulatory factors (e.g., <i>pflB</i>, <i>sarA</i>). Drug Affinity Responsive Target Stability (DARTS) experiments implicated icaB (a PNAG deacetylase) and phospholipid synthase SAOUHSC_01260 as its candidate targets. Furthermore, GSK2018682 could increase membrane permeability, depolarize, and enhance fluidity to disrupt the <i>S. aureus</i> membranes. Exogenous phospholipid supplementation markedly attenuated the antibacterial efficacy of GSK2018682 against <i>S. aureus</i>. Finally, GSK2018682 displayed a strong efficacy in murine models of MRSA infection. In summary, our findings establish GSK2018682 as a promising anti-<i>S. aureus</i> agent with dual antibacterial and antibiofilm activities, acting through interaction with membrane phospholipids to disrupt membrane integrity and offering a strategy against resistant <i>S. aureus</i> infections.