Pyrazole‐Derived Antibacterial Compounds Effectively Treat Methicillin‐Resistant Staphylococcus Aureus Infections by Inhibiting Aspartate Transcarbamoylase

ABSTRACT The persistent threat of methicillin‐resistant Staphylococcus aureus (MRSA) underscores the critical need for antimicrobials with novel mechanisms of action. Herein, we describe the discovery of Py‐27, a pyrazole‐derived compound exhibiting potent bactericidal activity against MRSA. A chemoproteomic approach identified aspartate transcarbamoylase (ATCase), a key enzyme in de novo pyrimidine synthesis, as the specific target of Py‐27. Biophysical and enzymatic assays confirmed its high‐affinity binding and inhibition of ATCase, while genetic knockout established ATCase as a druggable metabolic vulnerability in MRSA. Mechanistically, Py‐27 disrupts pyrimidine synthesis, thereby impairing DNA replication, perturbing metabolic homeostasis, and ultimately inducing lethal oxidative damage. Notably, Py‐27 eradicates mature biofilms while exhibiting a low resistance propensity and favorable toxicological profiles. In animal infection models, Py‐27 demonstrated superior in vivo efficacy compared to vancomycin and promoted infection resolution and tissue repair. This study demonstrates the potential of pyrazole‐amide compounds targeting ATCase for the treatment of MRSA infections.