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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.