Development of macrocyclic peptides as affinity probes

Lead Research Organisation: University of Oxford
Department Name: SABS IDC

Abstract

This project falls within the EPSRC 'chemical biology and biological chemistry' growth research area, and aims to develop macrocyclic peptides as affinity probes against enzymes which are important in epigenetic processes. Probes are molecules which selectively inhibit the function of an enzyme(s), and are developed to elucidate more about the biochemical role of the enzyme(s)1. An affinity probe is a probe with a reporter group, or the ability to add a reporter group after binding, often used to study interactions of the protein with other molecules (e.g. enzymes, DNA, etc.)2,3. Macrocyclic peptide probes offer advantages over small molecule probes for many reasons, including the potential to use them to target larger sites (including protein-protein interaction pockets) allowing targets traditionally thought of as "undruggable" using small molecules to be probed4. The larger surface area of peptides also facilitates high selectivity for the target protein over structurally similar proteins5. Initial work will focus on published macrocyclic peptides against histone lysine demethylases 4A-C (KDM4A-C), establishing methodology that will be applied to a set of cyclic peptides that will be screened against an enzyme implicated in DNA methylation/hydroxymethylation.
Small molecule probes against KDM4A-C, and enzymes key to DNA methylation/hydroxymethylation reported to date have had selectivity issues6-8, highlighting the need for a new approach to probe development in this area. A KDM4A-C selective cyclic peptide developed by Kawamura et al., CP29, will be further developed by linking it to a molecule that signals for protein degradation, potentially creating the first proteolysis targeting chimaera (PROTAC) against these targets. The PROTAC development will be in collaboration with GSK, Stevenage where they have expertise in the development of these probes. They will provide the section of the molecule that signals for degradation, which can be joined to CP2 in Oxford.
The second starter project will look for non-inhibitory KDM4A-C binding cyclic peptides from other peptides found in the CP2 cyclic peptide screen. Hydrogen/deuterium (H/D) exchange mass spectrometry (MS) can then be used to find out whether the peptides bind to the active site of KDM4A, or an allosteric site. In the large project, a cyclic peptide screen will be performed against either a ten-eleven translocation (TET) enzyme or DNA methyltransferase3A/B (Dnmt3A/B) to find binders. Techniques used to identify KDM4A-C allosteric binders will then be applied to the new cyclic peptides to find an allosteric site binding cyclic peptide that can be developed into an affinity cyclic peptide. The next generation of the DNA methylation/hydroxymethylation cyclic peptide will feature a handle to allow reporter groups to be attached, facilitating analysis of interacting proteins on these potentially important cancer targets10; a handle can also be used to develop the probe into a PROTAC inhibitor using methodology developed for CP2, allowing inhibitor studies to be performed.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R512333/1 01/10/2017 30/09/2021
1941447 Studentship EP/R512333/1 01/10/2017 30/09/2021 Grace Roper