| 研究課題/領域番号 |
16F16810
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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, 大学院理学系研究科(理学部), 教授 (00361668)
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| 研究分担者 |
WIEDMANN MAREIKE 東京大学, 大学院理学系研究科(理学部), 外国人特別研究員
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| 研究期間 (年度) |
2016-11-07 – 2019-03-31
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| キーワード | ペプチド / 特殊ペプチド / 翻訳 / 薬剤探索 |
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| 研究実績の概要 |
This project s to further develop and optimise the macrocyclic peptides targeting the enzyme iPGM in parasitic nematodes. This new drug that would be able to kill adult nematodes would be highly beneficial for human society. Cofactor independent phosphoglycerate mutase (iPGM) catalyses the transfer of a phosphate group from 3-phosphoglycerate to 2-phosphoglycerate via a catalytic serine residue in the nematodes. siRNA knockdowns of iPGM in nematodes are lethal at the embryonic and larval stages of development and also result in phenotypic deformations. The cyclic peptide discovered previously has an 8AA cyclic core and a 7AA tail and has broad-spectrum inhibitory activity against all iPGM orthologs. One issue with the identified peptide is its poor membrane permeability due to its high polarity and charge. Hence the new aim is to test which amino acids in Iperglycimide can be replaced with more hydrophobic ones to reduce its overall polarity. I first determined the acylation efficiency of 4-fluorophenylalanine using different incubation times. The correct incorporation of the unnatural amino acids into a peptide template was checked. Both N-chloroacetyl-D-Tyr and the 4-fluoro-phenylalanine were incorporated correctly into the peptide template sequence as verified by Maldi MS. A consecutive bead binding assay confirmed the binding capacity for Ce-iPGM protein. A clone was conducted and the desired cyclic peptide Iperglycimide via selection against Ce-iPGM protein was obtained.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
I have worked on an NNK mutational scan of all amino acids in Ce-2 for all other proteogenic AAs and also 4-fluorophenylalanine with the aim to reduce the overall polarity of the compound. I am currently preparing the library for Miseq sequencing. I have recently initiated exploring different targets that will be used during the screening assay. I am currently manually synthesizing Lipid II and variants containing only the amino acid tail fused to a PEG linker and fused to biotin. This will allow me to screen for binders using more established group methodology. A collaboration with Prof Schneider’s group at the University of Bonn was set up and they will provide us with the purified target lipid and variants in the near future.
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| 今後の研究の推進方策 |
Project 1: Future work involves running the MiSeq. This will be followed by data analysis and clone assays to verify the most interesting peptides. The most promising candidates will be synthesized on a larger scale, their binding affinities determined and then sent off to our collaborators at the NIH for further activity testing.
Project 2: Two different immobilisation methods will be explored to identify Lipid II binders: both C18 beads and Streptavidin beads will be used. The synthesised Lipid II variants only containing the amino acid tail of Lipid II will be immobilised on streptavidin beads and screened using the RaPID Assay. Future work will involve setting up the Streptavidin beads screening system using a more economical lipid alternative. Once the target lipid is obtained from the collaborators, it will be immobilized on C18 beads and screened using the RaPID assay. Once a set of binding ligands has been confirmed, these will be synthesised on a larger scale using standard Fmoc Solid-Phase Peptide Synthesis to be tested.
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