Development of chemical probes for DNA modifying enzymes

Lead Research Organisation: University of Oxford


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.


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Nowak RP (2021) First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53. in Journal of medicinal chemistry

Description Background
Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent dioxygenases and play a key role within the active DNA demethylation pathway by oxidising 5-methylcytosine, priming it for demethylation. As such, TETs are essential for gene expression regulation. Consequently, aberrant TET activity is often a hallmark of cancer, particularly leukaemias. Development of selective inhibitors and biochemical/structural characterisation of TETs is challenging due to lack of robust high-throughput biochemical assays and difficulties in isolating sufficient quantities of pure TET protein.

New knowledge generated
A library of cyclic peptide inhibitors identified in an mRNA display experiment against hTET1 has been synthesised and characterised against all 3 human TETs (hTET1-3), mouse Tet1 (mTet1), and Naegleria Tet1 (NgTet1), as well as histone lysine demethylases KDM5B and 6B.
The cyclic peptide with the best inhibitor profile, TiP1, has been selected for further characterisation - an alanine scan is being carried out to identify key residues required for binding to the target protein.
Significant work towards optimisation of hTET2 expression in E. coli and subsequent purification has been carried out to facilitate structural studies. hTET2 crystallisation optimisation is underway to enable co-crystallisation with inhibitors Co-crystallisation with both small molecule and cyclic peptide inhibitors would allow to rapidly develop the inhibitors using structure-guided design.
A series of cell-permeable small molecule inhibitors inhibiting hTET2 in the triple-digit nanomolar/single-digit micromolar region have been identified. Characterisation against other 2OG dioxygenases is underway to determine cross-family selectivity.
Exploitation Route The development of a potent, selective, and cell-permeable TET inhibitor (either cyclic peptide or small molecule) would allow further TET characterisation in a cellular context. This could potentially enable early target validation in a pharmaceutical context by using the developed inhibitor to disrupt TET activity in mammalian cell lines in a spatiotemporal fashion, mimicking a response to a drug molecule.
Cyclic peptide and small molecule inhibitor structure-activity relationship characterisation using biochemical assays and crystallography (co-crystallisation of TET2 with inhibitors) could potentially be used to expedite the discovery (computational docking) and lead optimisation (structure-guided design) of novel inhibitors with a core more suitable for medicinal chemistry.
Sectors Pharmaceuticals and Medical Biotechnology

Description Collaboration with Assoc Prof Dante Rotili (Sapienza University of Rome) on the development of TET dioxygenase inhibitors 
Organisation Sapienza University of Rome
Country Italy 
Sector Academic/University 
PI Contribution Design and screening of compounds against dioxygenases of interest.
Collaborator Contribution Design and chemical synthesis of compounds of interest.
Impact No outputs as of yet. Multi-disciplinary, including medicinal synthetic organic chemistry, biochemistry, chemical biology.
Start Year 2018
Description Epigenetics exhibit stall at the IF Oxford: The Oxford Science and Ideas Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Together with our research group, we developed and presented an interactive stall focusing on epigenetic control through DNA modification at the IF Oxford: The Oxford Science and Ideas Festival. Strong public engagement and interest was seen. We discussed science concepts that were hard to grasp with the guests, engaging in discussion with persons ranging from the general public to professionals from other areas of life science. Overall, the festival is a strong case for improving the communication between scientists and the general public.
Year(s) Of Engagement Activity 2019
Description Online talk on epigenetics for non-scientists 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact An online talk was prepared on the effect of epigenetics for Lithuanian audiences which has been viewed nearly 90 times, with some viewers getting in touch for further information.
Year(s) Of Engagement Activity 2020
Description University of Oxford Chemistry Department Open Door event 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Over 50 persons attended the Chemistry Open Day. Facilities and equipment were shown, and lots of chemistry/biology concepts were discussed, with common misconceptions being cleared up.
Year(s) Of Engagement Activity 2019