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The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically‐triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click‐to‐release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse‐electron‐demand Diels–Alder reaction between trans‐cyclooctene (TCO) and the amine‐functionalised tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d‐Ubi−Tz, a tetrazine functionalised with the bacterial binding peptide d‐Ubi29–41. While standard TCO did not improve toxicity against human proximal tubular kidney HK‐2 cells, the installation of an aspartic acid‐modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col−(TCO‐Asp)1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d‐Ubi−Tz in an in vivo infection model, whereas it remained inactive and non‐harmful without the chemical trigger. This study constitutes the first example of a systemically acting two‐component antibiotic with improved drug tolerability. [ABSTRACT FROM AUTHOR]

Summary: Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspFU (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspFU. IRSp53 colocalizes with EspFU and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspFU and N-WASP but requires Tir residues 454–463, previously shown to be essential for EspFU-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspFU in EHEC pedestal formation. [Copyright &y& Elsevier]

The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene (TCO) and the amine-functionalised tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi 29-41 . While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp) 1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

In order to render potent, but toxic antibiotics more selective, we have explored a novel conjugation strategy that includes drug accumulation followed by infection-triggered release of the drug. Bacterial targeting was achieved using a modified fragment of the human antimicrobial peptide ubiquicidin, as demonstrated by fluorophore-tagged variants. To limit the release of the effector colistin only to infection-related situations, we introduced a linker that was cleaved by neutrophil elastase (NE), an enzyme secreted by neutrophil granulocytes at infection sites. The linker carried an optimized sequence of amino acids that was required to assure sufficient cleavage efficiency. The antibacterial activity of five regioisomeric conjugates prepared by total synthesis was masked, but was released upon exposure to recombinant NE when the linker was attached to amino acids at the 1- or the 3-position of colistin. A proof-of-concept was achieved in co-cultures of primary human neutrophils and Escherichia coli that induced the secretion of NE, the release of free colistin, and an antibacterial efficacy that was equal to that of free colistin., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)