2020 Fiscal Year Annual Research Report
血栓形成の制御を目指した多数のジスルフィド結合を持つ天然物様環状ペプチドの開発
| Project/Area Number |
20J11284
|
|---|
| Research Institution | The University of Tokyo |
|---|
Principal Investigator |
LIU Wenyu 東京大学, 理学系研究科, 特別研究員(DC2)
|
|---|
| Project Period (FY) |
2020-04-24 – 2022-03-31
|
| Keywords | disulfide-rich peptides / in vitro selection |
|---|
| Outline of Annual Research Achievements |
This research aims at discovery of de novo disulfide-rich peptides (DRPs) with desired conformations and biological activities using in vitro selection. Since conformation of DRPs mainly depends on their disulfide connectivity, I intended to control the peptide structure by incorporating chemically protected cysteines during translation and stepwise deprotecting to form desired disulfide connectivity. I constructed 2 DRP libraries with bicyclic or tricyclic conformations and achieved in vitro discovery of target-binding DRPs against therapeutic targets. This work is the first trial of controlling peptide conformation via disulfide manipulation during in vitro translation and further application of this method will encourage discovery of more DRP based bioactive molecules.
|
| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
In the first year of my JSPS DC2 fellowship, I focused on ribosomally constructing a bicyclic DRP library with globular conformation using protected cysteines and successfully screened the library against mouse asialoglycoprotein receptor (mASGR) using in vitro selection. Since it’s our first time to control the complicated disulfide conformation during in vitro translation, the original plan that I set for the first year was to establish and optimize the selection system for bicyclic DRP library, however, even under the condition of reduced working time due to COVID-19, I’ve finished the real selection and successfully obtained a target binding bicyclic peptide with designed structure. Furthermore, I’m moving on to evaluation of the binding peptide with cell-internalization experiments.
|
| Strategy for Future Research Activity |
After successfully constructing and selecting the bicyclic library, I'm moving on to designing a constrained tricyclic DRP library with the desired knotted disulfide connectivity. In addition to Acm group, an orthogonal cysteine protecting group, phenylacetamidomethyl (Phacm), will be introduced to facilitate the stepwise deprotection and formation of 2 disulfide bonds. The library will be screened against therapeutic target proteins and the target-binding peptides will be chemically synthesized. Their disulfide connectivity and peptide conformation will be determined by LC-MS and their bioactivities will be evaluated by biological assays.
|
