Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

To demonstrate linker specificity for OmpT, we synthesized an activity probe 24, 29-33 with free linker peptides containing a fluorophore-quencher pair and detected OmpT activity in samples incubated with recombinant OmpT or live E. coli culture. Conversely, we observed no activity in samples containing the serine protease inhibitor, Aprotinin, which inhibits OmpT when present in micromolar concentrations 23 (Fig. 1C) . We observed similar cleavage activity using this linker 15 substrate when fully integrated into hairpin AMP drug-lock complexes, confirming that linker presentation within a constrained conformational state did not affect cleavage activity by OmpT (Fig. 1D) . To measure cytotoxicity of unlocked drug, we dosed bacteria with free AMP and observed significant reduction in colonies compared to untreated controls (blue bars) (Fig. 1E, F ). To confirm prodrug specificity, we synthesized AMP drug-lock complexes using linker peptides 20 specific for OmpT or Tobacco Etch Virus Protease (TEV), which exhibits orthogonal protease specificity 34 . We observed elimination of bacteria only in samples containing OmpT-specific AMP prodrugs (grey bars) or samples treated with both TEV and TEV-specific AMP prodrugs (red bars).