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submitted on 14.08.2020 and posted on 17.08.2020by Jonathan Boyce, Bobo Dang, Beatrice Ary, Quinn Edmondson, Charles Craik, William F. DeGrado, Ian Seiple
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.