Of all DNA damage forms, double-stranded breaks are the most genotoxic to the cell. Their repair produces concatenated DNA that must be cleaved before the cell divides in order to avoid mitotic catastrophe. MUS81-EME1 is a DNA repair endonuclease active in the cleavage of these DNA intermediates and it is this role that makes it a potentially interesting target in cancer therapy - sensitising cells towards chemo/radiotherapy. In order to investigate the behaviour of cells under MUS81-EME1 inhibition, and to explore potential synthetic lethalities, potent chemical probes must first be developed.

This research identified a modular, fluorogenic DNA reporter substrate design that provides a real-time kinetic readout of DNA repair activity. Of note, the modular design of the fluorogenic substrate provides instant access to the interrogation of an entire family of DNA repair nucleases. This enabled a highly optimised and efficient drug candidate screening assay (5 uL, 1536-format) that was amenable to total automation- capable of economically screening many thousands of compounds per hour. 

This poster describes the biochemical design, automation set-up, and computational screening approaches used to identify six ligand-efficient micromolar inhibitors of MUS81-EME1, as well as ongoing work for biophysical triage and structural studies to support rational medicinal chemistry and chemical probe optimisation.