Enantiomer-dependent biodistribution and exposure determine the in vivo behavior of a pleurocidin-derived antimicrobial peptide

ABSTRACT Antimicrobial peptides (AMPs) display rapid bactericidal activity in vitro , but have largely failed as systemically administered therapeutics, indicating that intrinsic potency alone does not predict in vivo efficacy. Here, we examine how pharmacokinetics, stereochemistry, and host context shape early in vivo activity using pleurocidin-derived peptides. The D -amino-acid analog D-P-KR rapidly eradicates priority bacterial pathogens within minutes in vitro , achieving this at low multiples of the minimum inhibitory concentration (MIC) for Staphylococcus aureus but requiring higher multiples for Gram-negative species. Near-infrared imaging of fluorescently labeled enantiomers reveals that stereochemistry strongly influences systemic behavior, with D-enantiomers showing reduced renal and hepatobiliary clearance and increased exposure following intravenous administration. Despite these favorable properties, systemic D-P-KR does not reduce pulmonary bacterial burden within 2 h of treatment in mouse models of S. aureus or Klebsiella pneumoniae pneumonia. In contrast, increasing exposure through short intravenous infusion produces a rapid, exposure-dependent reduction in pulmonary neutrophil recruitment in S. aureus infection, without corresponding early changes in bacterial burden. This effect is not observed in the disseminating K. pneumoniae model. These findings demonstrate a dissociation between rapid in vitro killing, early bacterial clearance, and host-response modulation, and show that early in vivo AMP activity is governed primarily by systemic exposure and infection context. More broadly, this work highlights the need to integrate pharmacokinetics, dosing strategy, and host response when evaluating antimicrobial peptides for systemic use. IMPORTANCE Antimicrobial peptides can kill bacteria far more rapidly than most conventional antibiotics, yet very few have succeeded as systemically administered drugs. This study shows that differences in peptide stereochemistry profoundly influence how antimicrobial peptides distribute within the body and how long effective exposure can be maintained. Our findings demonstrate that strong antibacterial activity in vitro does not necessarily translate into early bacterial clearance in vivo , but that delivery strategies can still affect host responses during infection. By linking peptide stereochemistry, systemic exposure, and biological outcomes, this work highlights why many antimicrobial peptides struggle to translate clinically and underscores the need to prioritize pharmacokinetics, formulation, and dosing strategy alongside antibacterial potency during antimicrobial peptide development.