Platform to Discover Protease-Activated Antibiotics and Application to Siderophore–Antibiotic Conjugates
- Jonathan H. BoyceJonathan H. BoyceDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesCardiovascular Research Institute, University of California, San Francisco, California 94158, United StatesMore by Jonathan H. Boyce,
- Bobo Dang*Bobo Dang*[email protected]Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, ChinaCenter for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, ChinaInstitute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, ChinaMore by Bobo Dang,
- Beatrice AryBeatrice AryDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesMore by Beatrice Ary,
- Quinn EdmondsonQuinn EdmondsonDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesMore by Quinn Edmondson,
- Charles S. CraikCharles S. CraikDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesMore by Charles S. Craik,
- William F. DeGrado*William F. DeGrado*[email protected]Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesCardiovascular Research Institute, University of California, San Francisco, California 94158, United StatesMore by William F. DeGrado, and
- Ian B. Seiple*Ian B. Seiple*[email protected]Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, United StatesCardiovascular Research Institute, University of California, San Francisco, California 94158, United StatesMore by Ian B. Seiple
Abstract
Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for 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 of E. coli 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. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (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 siderophore-facilitated 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.