New Genetically Engineered Derivatives of Antibacterial Darobac-tins Underpin their Potential for Antibiotic Development

Biosynthetic engineering of bi–cyclic darobactins, which selectively seal the lateral gate of the outer membrane pro-tein BamA, lead to highly active analogues which are up to 128–fold more potent against critical and clinically relevant Gram–negative pathogens compared to their native counterparts. Because of their excellent antibacterial activity, darobactins represent one of the most promising new antibiotic classes of the last decades. Here we present a series of structure-driven biosynthetic modifications of our current frontrunner, darobactin 22 (D22), to investigate modifica-tions at the understudied positions 2, 4 and 5 of the darobactin heptapeptide for their impact on bioactivity. Novel darobactins were found highly active against critical pathogens from the WHO priority list. Antibacterial activity data were corroborated by determination of the dissociation constants KD with BamA beta barrel. The most promising de-rivatives, D22 and D69, were subjected to ADMET profiling, showing high metabolic and plasma stability and low plasma protein binding. We further evaluated D22 and D69 for bioactivity against multidrug–resistant clinical isolates and found them to have low to submicromolar activity.