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Application of peptide screening technology for identification of substrates of the Jumonji-C histone demethylases

Periodic Reporting for period 2 - RAPID-KDM (Application of peptide screening technology for identification of substrates of the Jumonji-C histone demethylases)

Reporting period: 2017-10-01 to 2018-09-30

The functional human proteome is vastly expanded by post-translational modifications that modify protein function and gene expression in a time- and context-dependent manner. These are particularly important in the field of epigenetics – the study of changes in gene regulation not caused by changes in the underlying DNA sequence. Misregulation of these modifications correlates with a wide range of developmental disorders and disease pathologies, accounting for a high burden of disease and deaths in the EU. Enzymes that regulate post-translational modifications therefore represent key drug targets for both academics and the pharmaceutical industry.

To successfully manipulate these enzymes and marks for therapeutic benefit it is essential that we understand their roles within a cell. Despite their great importance, the full roles and regulation of these modifications and epigenetic enzymes remain poorly understood. Modifications of arginine residues remain especially understudied. The overall scientific objective of this project is thus to develop screening methodology, based on cutting-edge mRNA-display-based screening technology (the RaPID system) developed by the Suga laboratory (University of Tokyo), to identify enzyme substrates. Additionally, the work aims to develop inhibitors/peptide probes for these epigenetic enzymes, which will serve vital functions both to further elucidate the functions of these proteins within cells and also to act as drug leads.

To achieve these scientific aims, a major objective of this project is to provide the fellow with training in the state-of-the-art RaPID system and other advanced methodologies. Combined with the exposure to a different culture and language, and the opportunity to observe how research is conducted in another country, this fellowship will be invaluable in accelerating the fellow’s career trajectory on return to the EU.
The RaPID system allows incorporation of non-canonical amino acids into peptides. It has been expanded to allow incorporation of symmetric and asymmetrically dimethylated arginine into peptide sequences. Warhead libraries, allowing inclusion of these post-translational modifications in the selected peptides have also been designed and synthesised. Using these novel libraries and post-translational modification incorporations, peptide selections were carried out with the histone demethylase KDM6B. Unfortunately, further analysis revealed that the identified peptides were not substrates of KDM6B. It seemed likely that this is due to the peptides binding too tightly to be substrates for the JmjC KDMs, suggesting that in this context the proposed methodology might not be optimal for KDM substrate identification. However, screening studies with histone peptides, followed by kinetic and structural characterisation, revealed that a site on histone H1.4 lysine 26, is a comparably good substrate for the KDM4 subfamily of histone demethylases as the better studied histone H3K9 site and a better substrate than the H3K36 site. This work has been published in FEBS Letters.

To further pursue our original substrate hunting methodology, peptide selections have also been carried out with two other families of epigenetic proteins: PADI4 and a family of bromodomains. PADI4 is a histone arginine deiminase, that is an important rheumatoid arthritis target (a disease that affects around 1% of the population). Selections with PADI4 identified tight binding peptides that were also enzyme substrates – target arginine residues within the peptides could be converted to citrulline. Due to their very potent binding these peptides have been pursued as inhibitors of PADI4. In collaboration with colleagues at the University of Edinburgh they have been tested in cells and show cellular activity. Using scanning experiments, the lead peptide sequence has been further optimised. A non-inhibitory peptide has also been converted into a biotinylated pull-down reagent, which provides a powerful alternative to selective antibodies. This work has been presented at several conferences and is currently being prepared for open access publication. Finally, a set of bromodomains have been profiled. These proteins bind to acetylated lysine residues, and are currently being pursued as cancer treatment targets. Selections with these proteins identified potent binding peptides, though once again they appear not to represent cellularly relevant sequences. Structural characterisation by NMR and crystallography in collaboration with colleagues at the University of Sydney have revealed a wide range of binding modes that can be further exploited to achieve higher levels of selectivity than have previously been observed for small molecule inhibitors of these proteins. The results are currently being written up for publication.
The RaPID system is a cutting-edge technology for drug discovery not widely implemented in Europe. Through her two-year secondment to the University of Tokyo the fellow received extensive training in the use of this technology from its pioneer. She also received training in other complementary scientific techniques, such as advanced peptide synthesis and biophysical methods. She is now establishing the RaPID system in the UK, enhancing the research capabilities of the EU. Moreover, this advanced training will prove invaluable for her future career, allowing her to develop an independent research programme based around use of the RaPID system. The secondment to Japan gave her experience of how research is conducted in another country and has the opportunity to learn a new language and about another culture.

Overall the fellowship has thus trained the fellow to become a female European research leader with a global outlook on research. From this position, she can act as a role model for younger female scientists and school children, helping to address the imbalance of women in the scientific workforce. Through a range of outreach activities, such as giving lab tours, she has already begun to address this. Additionally, whilst in Japan the fellow spoke at events promoting fellowships and PhDs in Europe, allowing her to encourage the movement of skilled researchers to the EU.

The RaPID system has been further extended to allow incorporation of amino acids that cannot be stereoselectively incorporated using other reprogramming technologies. Incorporation of post-translationally modified amino acids into selections helped to achieve high potency for peptides selected against bromodomains, and can be used more widely by other researchers interested in post-translational modifications. Experiments with histone H1 peptides have also progressed our understanding of the substrate specificity of KDMs, which will be important for our analysis of cell-based studies. Further, we have identified highly potent inhibitors of PADI4, which, have the potential to lead to a novel treatment for rheumatoid arthritis, from which a large number of patients in the EU and worldwide could benefit. Downstream development of the bromodomain binders has the potential to provide new drugs for the treatment of cancer. Together our results will be of interest to a very wide range of scientists including other academic researchers in the field of epigenetics and beyond, and major biotechnology and pharmaceutical companies studying JmjC KDMs, arginine deiminases and bromodomains as drug targets.
RaPID selections for identification of substrates and inhibitors