Category: Chemical Biology

1185-A - Modular, Fully-Automated HTS Assay & Hit Identification for DNA Repair Nucleases

Monday, February 5, 2018
2:00 PM - 3:00 PM

Intrinsic and extrinsic sources of DNA damage are ubiquitous and pose a threat to the integrity of the genetic information stored in our cells; the decline in information quality through damage and imperfect replication underpins cancer, aging and many degenerative disorders. Necessarily, numerous repair pathways have evolved. Of all DNA damage forms, double-stranded breaks are the most genotoxic to the cell. Their repair produces concatenated DNA (Holliday junctions) that must be cleaved before the cell divides in order to avoid mitotic catastrophe. MUS81-EME1 is a structure-selective DNA repair endonuclease active in the cleavage of these DNA intermediates and it is this important 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, and so appropriate high-throughput screening technologies were developed.

This research has 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/podium talk] will describe the biochemical design, automation set-up, and computational screening approaches used to identify six highly 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. This interdisciplinary work spans chemical biology, laboratory automation and drug discovery and will be of interest to those in early stage drug discovery, automation and screening technologies.

Thomas Fleming

PhD Chemical Biology
University of Oxford
Oxford, England, United Kingdom

I am a chemical biology PhD scientist and Fellow of the Royal Commission of 1851, deeply interested in the Chemistry-Biology interface. I am currently investigating DNA damage & repair in the 'Synthesis for Biology & Medicine Centre for Doctoral Training' (SMB CDT) at the University of Oxford. I aim to find new drugs to restore efficacy of chemotherapy for hard-to-treat cancers, where the development of resistance to the anti-cancer drugs leave patients without other treatment options. My research involves the use of high-throughput integrated robotics and miniaturised assay technologies. This research addresses a serious unmet clinical need, and has major potential societal impact.