2014 Fiscal Year Annual Research Report
プロリル－４－ヒドロキシダーゼをアイソフォーム選択的に阻害する大環状化合物の発見
| Project/Area Number |
14F04065
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| Research Institution | The University of Tokyo |
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Principal Investigator |
菅 裕明 東京大学, 理学(系)研究科(研究院), 教授 (00361668)
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| Co-Investigator(Kenkyū-buntansha) |
LOIK Nikita 東京大学, 理学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2014-04-25 – 2016-03-31
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| Keywords | 遺伝記号 / 特殊ペプチド / チオオキシカルボン酸カルボン酸 / tRNAアルシ化 / 翻訳 |
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| Outline of Annual Research Achievements |
Since beginning work on this project, I have successfully synthesised the hydroxamic analog of α-aminosuberic acid, which was successfully incorporated into the nascent peptide chain in vitro using dFx flexizyme technology. This allowed me to use it as a part of cyclic-peptide-inhibitor. To identify the binding peptide sequences from both ‘warhead’ and ‘no-warhead’ libraries, two selections were carried out against PHD2 enzyme. For both libraries, the 20 most abundant cyclic peptide sequences were identified. Gratifyingly, the selected cyclic peptides demonstrated high consensus in their sequences.
In addition, an alternative method for the assessment of cyclic peptide cell-penetration was developed. The method was tested using cyclosporine and showed promising results with the ability to identify cyclosporine in cyclosporine-treated cell digest at low nano-molar level.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
This work required learning new techniques and background information in the bio-chemical field. Successful completion of the first stage of my project signifies the fulfilment of the learning objective despite a very steep learning curve.
To date synthesis and test of the ‘warhead’ ― the major milestone of this project ― was successfully achieved. It was also possible to perform selection against stable PHD2 isoform using two different libraries, which provided potential cyclic-peptide inhibitors. In addition, I engaged in the development of the alternative assays for cyclic peptide cell penetration and histone modification analysis, thus contributing to the repertoire of methods available to the laboratory.
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| Strategy for Future Research Activity |
To finalise selection of the inhibitors against PHD2, the identified clones will be tested for binding individually. The successful binding proteins will be re-synthesised using solid phase synthesis and their inhibitory activity will be tested individually against PHD2. For the successful inhibitors, cell permeability will be tested using both conventional fluorescein labelling and an LC-MS based test.
To finalise the LC-MS based method for the assessment of peptides’ cell permeability, further testing using in-house synthesised cyclic peptides will be carried out. The results of the LC-MS based test will be compared against fluorescein labelling.
I will be also attending the EMBO Conference Series: Chromatin and Epigenetics, which ― I expect ―provides me with further insight into the histone methylation as a mechanism of gene activation regulation and enables wider collaboration on this project.
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