Authors

S L Oates²; J R Sayers²; H Price¹;  ¹ Keele University;  ² The University of Sheffield

Discussion

Emerging drug resistance in kinetoplastid parasites is increasing the burden on the global health system. It is therefore essential to identify and characterise novel therapeutic targets. Flap endonucleases are involved in DNA replication and repair. These enzymes have been shown to be essential in other organisms including mice and bacteria. The human homologue of the enzyme is up-regulated in certain cancers, and has potential as a target for anti-cancer therapies. In the current study we analysed the potential of the Trypanosoma brucei flap endonuclease as a therapeutic target. A wild-type and a mutated variant of the flap endonuclease were over-expressed in E. coli. A comparative analysis was performed on the recombinant proteins using a DNA cleavage assay, based on Förster resonance energy transfer (FRET), a bio-layer interferometry-based DNA binding assay and circular dichroism. These analyses showed that the mutation rendered the enzyme catalytically inert, whereas structural integrity and DNA binding were similar to the wild-type. Over-expression of both the wild-type and variant genes were analysed in the T. brucei parasite. The catalytically inert protein had a significantly detrimental effect on cell growth, and morphological changes were observed 72 hours post-induction. However, no effect on cell cycle progression was observed after 72 hours. A known flap endonuclease inhibitor, myricetin, was assessed for its ability to target the T. brucei flap endonuclease.The ability of myricetin to interact with the T. brucei flap endonuclease was assessed in silico, and inhibitory effects analysed in vitro. Inhibition of the T. brucei FEN correlated with cell death in vivo. Further work is in progress to determine if cell death is due to FEN1: myricetin interaction.