| Project/Area Number |
23KF0020
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund |
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| Section | 外国 |
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| Review Section |
Basic Section 37020:Chemistry and chemical methodology of biomolecules-related
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| Research Institution | The University of Tokyo |
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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) |
LI CHOI YI 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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| Project Period (FY) |
2023-04-25 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2024: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2023: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Disulfide bond / Cysteine / RaPID system |
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| Outline of Research at the Start |
By the end of this Fellowship, we expect the strategy to construct cyclic DRP-based libraries for in vitro selection can be well established and published so that this strategy can be used by other research groups for drug discovery.
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| Outline of Annual Research Achievements |
This project addresses the need to develop a novel in vitro selection platform capable of displaying cyclic DRP-based libraries. I used a thioester-closed cyclic peptide as the initial scaffold to produce cyclic disulfide-rich peptides (DRPs) using the RaPID system and a cysteine protection and deprotection strategy. I have successfully identified the optimized conditions (buffer, pH value, etc.) and the suitable cysteine sidechain protecting group (phacm) for use in the RaPID system to efficiently produce DRPs. Using these optimized conditions, an mRNA display library consisting of cyclic DRPs that contain only one disulfide bond has been successfully constructed and used to perform affinity selection against gasdermin D, a novel therapeutic target for autoimmune diseases. I have successfully identified 18 DRPs from the mRNA display library that show binding activity towards gasdermin D. These promising peptide binders have been synthesised and purified for further evaluation.
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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
The identified cyclic DRP-based compounds for gasdermin D have been synthesized and are currently being evaluated. Surface Plasmon Resonance (SPR) is employed to measure the equilibrium dissociation constant (KD) of these compounds towards gasdermin D. The SPR results showed that all these compounds exhibit binding activity towards gasdermin D. Additionally, two of these compounds display a low KD value, indicating strong binding affinity for gasdermin D. Concurrently, the in vitro gasdermin D cleavage experiment is being used to further verify the binding affinity of these compounds towards GSDMD. The cleavage assay results are consistent with the SPR findings. Those two compounds showing low KD values prevent gasdermin D from being cleaved by enzymes. Based on these results, we can conclude that we have identified two DRP-based compounds that are promising gasdermin D binders. More evaluations are ongoing.
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| Strategy for Future Research Activity |
Since the bicyclic DRP-based library has proven successful, to further advance this project, I will construct a multicyclic DRP-based library. Cyclic DRPs containing a cysteine knot (three disulfide bonds) have garnered considerable attention recently as a template for engineering protease inhibitors. I will utilize the strategy developed in 2023 to construct a cyclic MCoTI-based library for in vitro selection against two novel therapeutic targets: matriptase-2 and TMPRSS2. Matriptase-2 has been identified as a promising therapeutic target for iron overload diseases, and TMPRSS2 has been linked to COVID-19 disease conditions. The identified compounds will be further evaluated using in vivo experiments in collaboration with our partners.
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