Periodic Reporting for period 2 - RAPID-KDM (Application of peptide screening technology for identification of substrates of the Jumonji-C histone demethylases)
Okres sprawozdawczy: 2017-10-01 do 2018-09-30
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.
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.
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.