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GENERATION OF A NEW AMINOCOUMARIN ANTIBIOTIC CONTAINING A CATECHOL STRUCTURE FOR IMPROVED UPTAKE ACROSS THE OUTER MEMBRANE Silke Alt, Nadja Burkard, Andreas Kulik, and Lutz Heide University of Tübingen, Pharmazeutical Institute, Auf der Morgenstelle 8, 72076 Tübingen, Germany, [email protected] Aminocoumarin antibiotics like clorobiocin and novobiocin produced by different Streptomyces strains are potent inhibitors of DNA gyrase. Although novobiocin has been licensed for clinical use in human infections with Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus strains, the clinical application of these antibiotics remains restricted. One reason is their low activity against Gramnegative pathogens. Therefore, it is of interest to test whether structurally modified aminocoumarin derivatives are able to overcome the limitations. One potentially powerful approach could be a Trojan-horse strategy using the iron transport abilities of catechol siderophores in order to carry drugs with catechol motifs into Gramnegative cells. To mimic the siderophore structure, we decided to introduce a catechol moiety - 3,4-dihydroxybenzoic acid (DHBA) - into clorobiocin instead of the genuine prenylated 4-hydroxybenzoyl moiety (ring A) by metabolic engineering. We first confirmed in vitro that 3,4-DHBA is accepted as substrate by the amide synthetase encoded in the clorobiocin gene cluster. Then, a modified clorobiocin gene cluster that lacks an essential gene of the biosynthesis of the genuine ring A was expressed heterologously in Streptomyces coelicolor M512, together with an expression plasmid containing synthetic genes for 3,4-DHBA biosynthesis. We detected the production of a new clorobiocin derivative with the desired catechol moiety and we confirmed the structure by LC-MS and NMR. In addition, we tested the inhibitory activity of the new compound toward DNA gyrase and topoisomerase IV in vitro as well as its antibacterial activity against different Escherichia coli mutant strains. The new clorobiocin derivative showed 5-fold better inhibitory activity against E. coli DNA gyrase than clorobiocin and also its antibacterial activity determined in agar diffusion tests was higher. Experiments demonstrated that its influx dependents on intact siderophore transporters. These results demonstrate the viability of this strategy to obtain improved aminocoumarins, and gives further insight into the structure-activity relationship of aminocoumarins relating both to their transport across the cell membranes and to their interaction with the target.