2017 Fiscal Year Annual Research Report
Project/Area Number |
16F16810
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Research Institution | The University of Tokyo |
Principal Investigator |
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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Co-Investigator(Kenkyū-buntansha) |
WIEDMANN MAREIKE 東京大学, 大学院理学系研究科, 外国人特別研究員
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Project Period (FY) |
2016-11-07 – 2019-03-31
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Keywords | RaPID System / mRNA display / quorum sensing / macrocyclic peptide / infectious diseases |
Outline of Annual Research Achievements |
Project 1 is to further develop and optimise a macrocyclic peptide, that a recent PhD graduate from the group, developed. It targets the enzyme iPGM in parasitic nematodes. 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. A DNA library was designed and synthesized that would yield an NNK at every amino acid position and hence result in point mutations along the entire peptide template. Through one selection cycle we were able to compare the relative binding affinities of all single mutants of the identified hit peptide. I next set out to design and synthesise different peptide analogues to improve the pharmacokinetic properties of the peptide. This was achieved by replacing amino acids that were charged and polar and by truncating the peptide tail. All synthesised peptides were HPLC purified and tested by my collaborators. Project 2 aims to discover novel binders of a membrane protein involved in quorum sensing of bacteria. The discovery of both agonists and antagonists would be desirable. We obtained sufficient quantities of the target protein from another collaborator and conducted a RaPID screen. Four novel macrocyclic peptides were discovered that were able to bind the membrane protein. Preliminary results suggest that two of the peptides are a partial agonist and the other an antagonist. The evaluation of the remaining two peptides is still outstanding.
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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
Project 1: Current work involved the further design and synthesis of peptide analogues and their in vitro testing. I am currently combining different single mutations into the same peptide and identifying suitable combinations. We are able to replace several tyrosine residues by phenylalanine or 4F-phenylalanine amino acids and I have also shown that we can replace parts of the peptidic tail by a non-peptidic linker. This should lead to an improved metabolic stability and an enhanced passive cell permeability of the resulting peptides. My collaborators are currently developing novel in vitro assays to test the peptides. Project 2: After the successful identification of peptide binders of a membrane protein that is immobilised via a nanodisc, I am now involved in screening a further membrane protein immobilised on a nanodisc. The protein is also involved in the quorum sensing of bacteria and both the identification of activators as well as deactivators would be desirable. I am also using the RaPID technology developed in the Suga lab to obtain binders followed by MiSeq identification. My collaborators are also developing a new in vitro assay to further characterise the peptide hits.
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Strategy for Future Research Activity |
Future work involves identifying a peptide that combines all favourable modifications that confer favourable pharmacokinetic properties ie improved metabolic stability, acceptable solubility and most importantly cell permeability (whether passive or active). Once a suitable peptide is identified the reaction synthesis will be scaled up for further in vitro and then also in vivo studies. The peptide will first be tested in cell-based assays and then tested on nematode (worm) cultures. My collaborators are currently developing an assay that may allow the insertion of the macrocyclic peptide into a bacterial host, which will serve as a delivery method to the nematodes that feed on bacteria. For Project 2 once the already discovered macrocyclic peptide hit compounds are fully characterised I aim to also run an NNK mutational scan on the peptides to identify single mutations that improve binding affinity and also improve pharmacokinetic properties. With the new membrane protein target, I will run the RaPID selection to identify novel binders. Binding will then be confirmed by conducting a clone assay and by Fmoc SPPS of the peptides. Activity will then be confirmed by my collaborators before progressing with the project. I am about to start a 3rd project. It is also in the infectious disease’s area. The new target protein is important in the biofilm formation of certain bacterial strains. We will be looking to identify macrocyclic peptides that are able to disperse biofilm formation. Once I have obtained the target protein, I will run a RaPID selection against the protein.
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Research Products
(3 results)