Development of chemical probes for DNA modifying enzymes

This project falls within the EPSRC Chemical Biology and Biological Chemistry research area.
Epigenetic modifying enzymes are capable of relaying chemical information through various mechanisms without altering the base DNA sequence. The modifications occur on either DNA or histones, and often result in changes of protein expression levels. These temporal and spatial changes in protein expression levels are hugely important for determining cell fate, as well as processes which are fundamental in mammalian development. A particular modification of interest to us is the (de)methylation of DNA, carried out by enzymes such as DNA methyltransferases (DNMTs), lysine demethylases (KDMs), and ten-eleven translocation enzymes (TETs). Given the importance of fluctuations of DNA methylation levels, obtaining chemical tools that can selectively inhibit a particular target is crucial for evaluating the functions of the enzymes. As such, our goal in this project is to develop highly selective enzyme inhibitors. In particular, we will focus on cyclic peptide inhibitors, which are known to have strong binding affinity and excellent target specificity, particularly over structurally similar proteins, and have been previously successfully employed within our group. Our initial investigations will focus on the human TET enzymes, for which no selective inhibitors have been reported to date. Developing selective inhibitors for this particular family of enzymes is extremely challenging, as they are part of a large superfamily of Fe(II)-dependent dioxygenases, all of which have highly conserved active sites. While broad-range inhibitors of these enzymes are available, the only work towards a selective inhibitor has been reported by our group, and a sufficiently selective compound has yet to be identified. We will design our inhibitors based on pre-existing structure-activity relationship data and synthesise them using standard solid phase peptide synthesis protocols. In parallel, we plan to optimise protein production of the target enzymes (human TET1, TET2, and TET3) to allow their production in sufficient quantities, followed by their biochemical and structural characterisation (enzyme kinetics, X-ray crystallography, etc.) to broaden our understanding of the enzymes and their active sites. The compounds will be tested against the enzymes using established enzyme assays, and the results will be utilised to design new iterations of synthetic targets. When a sufficiently potent and selective compound is identified, optimisation to improve its physiochemical properties, particularly solubility and cell permeability, will be carried out by introducing unnatural amino acids. Cell-based assays will be employed to evaluate the activity of the compounds in cells. In summary, we will be focusing on the development of target-selective chemical probes, as well as production of active constructs of the desired enzymes and their biochemical characterisation, all of which should enable us to ask questions about the function and activity of the targets.