title: Mechanistic studies on the application of DNA aptamers as inhibitors of 2-oxoglutarate-dependent oxygenases.
abstract: The Escherichia coli (E. coli) AlkB protein and its functional human homologues belong to a subfamily of 2-oxoglutarate (2OG) dependent oxygenases (2OG oxygenases for simplicity) that enable the repair of cytotoxic methylation damage in nucleic acids and that catalyze t-RNA oxidations. DNA alkylation is a major mechanism of action for cytotoxic anticancer drugs. Thus, the inhibition of oxidative demethylation, catalyzed by these enzymes, has the potential to improve the efficacy of chemotherapies. Here we report that oligonucleotide aptamers constitute a new class of potent inhibitors of 2OG oxygenases. DNA aptamers can selectively bind to AlkB, with nanomolar affinity, and efficiently inhibit catalysis. The mechanism of inhibition was studied by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Inhibition constants of the aptamers were determined and shown to correlate well with K(d) values. The results of kinetic analyses imply that the aptamers bind AlkB away from the active site. Our findings should stimulate the development of oligonucleotide aptamers for human homologues of AlkB and further their study as potential enhancers of chemotherapy efficiency.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, M3J 1P3, Canada.
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Krylova
authors.forename: Vasilij
authors.initials: V
authors.lastname: Koshkin
authors.forename: Eleanor
authors.initials: E
authors.lastname: Bagg
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D000600:Amino Acids, Dicarboxylic / Q000737:chemistry; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D055162:Biocatalysis; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D057075:Enzyme Assays; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry; D006801:Humans; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D000600:Amino Acids, Dicarboxylic; D052157:Aptamers, Nucleotide; D029968:Escherichia coli Proteins; D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D004247:DNA; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli; C040947:oxalylglycine