Chemically tailored anionic antibiotic adjuvants targeting divalent cations to overcome carbapenem resistance in gram-negative bacteria.

No clinically available antibiotic adjuvants can overcome metallo-β-lactamase (MBL) resistance in carbapenem-resistant Gram-negative ESKAPE pathogens. Divalent cations in MBL and quasicrystalline lipopolysaccharides (LPS) are key determinants of acquired and intrinsic resistance in these bacteria. Chemically depriving these cations would block multiple resistance pathways simultaneously and restore carbapenem susceptibility. Here, we chemically engineer a class of anionic adjuvants to tailor their interactions with MBL, enhancing the enzymatic inhibition by up to ~5885-fold compared to parent compounds. An orthogonal screen identifies the tiopronin-engineered anionic adjuvant (TINA), which achieves complete rescue of all tested carbapenems against diverse New Delhi metallo-β-lactamase-1-producing Gram-negative ESKAPE bacteria. TINA targets both MBL and LPS, depriving divalent cations to inactivate MBL and damage membrane collaboratively while also preventing resistance evolution. Moreover, TINA's anionic nature minimizes serum adsorption, enabling safe and effective in vivo treatment of bacterial pneumonia. This work provides an innovative chemical biology strategy leveraging anionic materials to combat superbugs globally.