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

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.