Data underlying the publication: Discovery of isokurarinone as an ATCase-Engaging Lead with potent activity against methicillin-resistant Staphylococcus aureus

The global proliferation of methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) persists as a significant contributor to challenging infections, highlighting the urgent necessity for therapies that utilize novel mechanisms. Aspartate transcarbamoylase (ATCase), which catalyzes the initial committed step of <em>de novo </em>pyrimidine biosynthesis, represents a promising metabolic target with potential relevance to MRSA fitness and persistence. Through structure-based virtual screening and experimental validation, we identified the flavonoid isokurarinone as a compound targeting ATCase, demonstrating potent anti-MRSA activity. Docking and molecular dynamics simulations, along with surface plasmon resonance and differential scanning fluorimetry, provided evidence for direct binding, which was accompanied by a reduction in ATCase activity and a decrease in <em>pyrB</em> expression. Non-targeted metabolomics revealed a disruption of pyrimidine nucleotide homeostasis, coinciding with impaired membrane integrity, reduced proton motive force, decreased intracellular ATP levels, and increased oxidative stress. Isokurarinone also inhibited biofilm formation and altered the expression of virulence-associated genes. In a murine model of MRSA-infected wounds, isokurarinone accelerated wound closure, reduced bacterial burden, and attenuated local inflammatory mediators. Collectively, these findings support the notion of ATCase as a metabolism-guided target for MRSA and nominate isokurarinone as a promising lead scaffold for therapeutic development.