Coupled Hydrogen-Bond–Electrostatic Recognition of Phosphatidylglycerol Drives the Design of Resistance-Suppressing Miniature Peptidomimetics

The emergence of multidrug-resistant (MDR) pathogens has urged us to find new antimicrobial strategies. Phosphatidylglycerol (PG) is an attractive bacterial-specific lipid target but is targeted by only one clinical agent, daptomycin. Yet daptomycin, like most reported PG binders, binds PG through an imprecise hydrophobic-electrostatic mode, necessitating a relatively large molecular size. This requirement, together with its strict Ca²⁺ dependence, significantly limits its efficacy. Here, we report <i>bis</i>-pyridinium amides (<b>BisPA</b>s), a rationally designed class of small molecules capable of precisely recognizing PG through amide-diol hydrogen bonding coupled with pyridinium-phosphate anionic-π interaction, independent of environmental conditions such as Ca²⁺. The lead compound, <b>BisPA14</b>, with ∼one-third the molecular weight of daptomycin, exhibits comparable PG-binding affinity, with <i>K</i>_d(<b>BisPA14</b>) = 1.4 × 10^-6 M versus <i>K</i>_d(daptomycin-Ca²⁺) = 0.9 × 10^-6 M. <b>BisPA14</b> disrupts PG self-assembly and membrane integrity and simultaneously engages bacterial DNA as a secondary intracellular target. This dual-targeting mechanism enables <b>BisPA14</b> to eradicate proliferating, tolerant, and persister bacterial populations while suppressing resistance evolution. It remains active in serum-containing environments, protects host cells from bacterial damage, and demonstrates excellent biocompatibility and strong therapeutic efficacy in intraperitoneal, pulmonary, and bloodstream methicillin-resistant <i>Staphylococcus aureus</i> infection models. As a synthetically accessible small molecule that functionally mimics and improves upon daptomycin's lipid-targeting mechanism, this work establishes a secondary-bonding-driven PG-recognition paradigm for combating MDR bacterial infections.