Aurachin D

The natural product aurachin D is a farnesylated quinolone alkaloid, which is known to possess activity against the causative agent of malaria, Plasmodium spp. In this study, we show that aurachin D inhibits other parasitic protozoa as well. While aurachin D had only a modest effect on Trypanosoma brucei rhodesiense, two other trypanosomatids, T. cruzi and Leishmania donovani, were killed at low micromolar and nanomolar concentrations, respectively, in an in vitro assay. The determined IC50 values of aurachin D were even lower than those of the reference drugs benznidazole and miltefosine. Due to these promising results, we set out to explore the impact of structural modifications on the bioactivity of this natural product. In order to generate aurachin D derivatives with varying substituents at the C-2, C-6 and C-7 position of the quinolone ring system, we resorted to whole-cell biotransformation using a recombinant Escherichia coli strain capable of aurachin-type prenylations. Quinolone precursor molecules featuring methyl, methoxy and halogen groups were fed to this E. coli strain, which converted the substrates into the desired analogs. None of the generated derivatives exhibited improved antiprotozoal properties in comparison to aurachin D. Obviously, the naturally occurring aurachin D features already a privileged structure, especially for the inhibition of the causative agent of visceral leishmaniasis.

A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 μM).

Aurachin D is a potent inhibitor of cytochrome bd oxidases, which are potential targets in the treatment of infectious diseases. In this study, our aim was to improve the biocatalytic production of aurachin D from a quinolone precursor molecule with recombinant Escherichia coli cells expressing the biosynthesis enzyme AuaA. In order to achieve a high-level production of this membrane-bound farnesyltransferase in E. coli, the expression of the auaA gene was translationally coupled to an upstream cistron in accordance with a bicistronic design (BCD) strategy. Screening of various BCD elements led to the identification of optimized auaA expression cassettes, which increased the aurachin D titer in E. coli up to 29-fold in comparison to T7-mediated expression. This titer could be further raised by codon optimization of auaA and by introducing the mevalonate pathway into the production strain. The latter measure was intended to improve the availability of farnesyl pyrophosphate, which is needed as a cosubstrate for the AuaA-catalyzed reaction. In sum, the described efforts resulted in a strain producing aurachin D with a titer that is 424 times higher than that obtained with the original, non-optimized expression host.