Biological and Medicinal Chemistry

Platform to Discover Protease-Activated Antibiotics and Application to Siderophore-Antibiotic Conjugates



Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkersfor prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads without an attached peptide “scar”. We next designed conjugates consisting of: 1) an N-terminal siderophore to facilitate uptake; 2) a protease-cleavable linker; 3) an amine-containing antibiotic. Using this strategy, we converted daptomycin – which by itself is active only against Gram-positive bacteria – into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophorefacilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform’s utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

Version notes

We changed one sentence in the revised version. Original sentence: “Proteolysis of conjugate 9 restores the activity of solithromycin in three ESKAPEE pathogens (E. coli, E. aerogenes, and K. pneumoniae) and in A. nosocomialis.” was changed to revised sentence: “Solithromycin conjugate 9 has comparable activity to solithromycin in three ESKAPEE pathogens (E. coli, E. aerogenes, and K. pneumoniae) and in A. nosocomialis.” on page 7, last paragraph. No changes were made to the Supporting Information.


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Supplementary material

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Supporting Information Boyce et al