Engineering a bacterial biosynthetic pathway for the synthesis of novel anticancer agents Dries De Ruysscher 1 , Eveline Vriens 1 , Evi Gorrens 1 , Neeve Marien 1 , Eveline Lescrinier 2 , Wim De Borggraeve 3 and Joleen Masschelein 1 1 KU Leuven - VIB Center for Microbiology, Belgium, 2 Medicinal Chemistry, Rega Institute-KU Leuven, Belgium, 3 Molecular Design and Synthesis, Chemistry Department, KU Leuven, Belgium The oximidines are hybrid polyketide-nonribosomal peptide natural products with potent and selective anticancer activity that have been identified as metabolites of Pseudomonas spp. The oximidines have a characteristic benzolactone enamide scaffold which has been proven to be a key structural feature for the selective inhibition of mammalian vacuolar (V-)ATPases, crucial enzymes for a broad variety of cellular processes. 1 As certain isoforms of these ATP-driven proton pumps are upregulated and mislocalised to the plasma membrane in cancer cells, they represent promising targets for the development of isoform-specific (chemo)therapeutics. 2 Oximidine biosynthesis involves several unusual features, including in-cis oxime formation, two mysterious isomerisation events and a highly unusual mechanism of chain release. In this work, detailed in vivo mutagenesis and in vitro biochemical assays were performed to elucidate the catalytic mechanisms of the enzymes responsible for these highly unusual transformations. In addition, novel oximidine derivatives were identified from wildtype Pseudomonas cultures and structurally characterized using a combination of 2D NMR spectroscopy and Mosher ester derivatisation. This combined structural and functional knowledge will further guide us towards generating improved anticancer agents by engineering the pathway or by chemically modifying the isolated oximidines via a structure-based drug design approach. References
1. Collins, M. P. and Forgac, M. Biophys. Acta Biomembr. 2020, 1862 , 183341.Oot, 2. R.A., Yao, Y., Manolson, M. F. and Wilkens, S. Biol. Chem. 2021, 297 , 100964.
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