| 研究課題/領域番号 |
14F04065
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| 研究機関 | 東京大学 |
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研究代表者 |
菅 裕明 東京大学, 理学(系)研究科(研究院), 教授 (00361668)
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| 研究分担者 |
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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| 研究期間 (年度) |
2014-04-25 – 2016-03-31
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| キーワード | 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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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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| 今後の研究の推進方策 |
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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