Crisaborole resensitizes KPC-producing Gram-negative bacteria to carbapenems

The global spread of carbapenem-resistant Enterobacteriaceae (CRE), primarily driven by the dissemination of carbapenemases, poses an existential threat to public health. Among these enzymes, Klebsiella pneumoniae carbapenemase (KPC) represents one of the most prevalent and clinically significant resistance determinants worldwide. In this study, we developed a PBP-based ELISA-like screening assay that exploits the differential recognition of intact meropenem and its KPC-mediated hydrolysis products by penicillin-binding protein. Using this assay, more than 300 FDA-approved drugs were screened, and crisaborole, an FDA-approved benzoxaborole compound, was identified as a potential KPC inhibitor. Checkerboard assays demonstrated synergistic activity between crisaborole and MEM against KPC-producing clinical isolates, with fractional inhibitory concentration index (FICI) values ranging from 0.19 to 0.50. Time-kill studies confirmed that the combination significantly reduced bacterial counts by >4 log₁₀ CFU/mL compared with MEM monotherapy. Notably, the synergistic effect was strictly dependent on the presence of the blaKPC gene; no activity was observed against strains harboring blaNDM, blaIMP, or blaOXA genes. Combined disc tests further validated that crisaborole directly inhibits KPC-mediated MEM hydrolysis. Molecular docking revealed that crisaborole binds to the active pocket of KPC through a covalent interaction with Thr237 and forms hydrogen bonds with Thr235, Ser130, and Asn132, with a binding free energy of -44.45 kcal/mol. These findings establish crisaborole as a promising repurposed antibiotic adjuvant for overcoming KPC-mediated carbapenem resistance and provide a rational foundation for further in vivo validation and clinical development.