2013 Fiscal Year Research-status Report
βーグルカン構造に基づく新規ワクチンアジュバントの開発
| Project/Area Number |
24710242
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| Research Institution | Hokkaido University |
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Principal Investigator |
ファイナ ガルシア・マーティン 北海道大学, 先端生命科学研究科(研究院), 助教 (30537080)
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| Keywords | β-glucan / adjuvant / solid-phase synthesis / Dectin-1 / glycan microarray / binding assay |
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| Research Abstract |
The objective of the research project is the obtaining of 1,3-β-glucan and further biological studies. The hypothesis is that 1,3-β-glucan as ligands of Dectin-1 can stimulate the immune system and the feasibility to be used as adjuvants.
During FY2013, optimization of solid-phase method was performed and still ongoing. This was a very critical step as many polymers for solid phase were used but under glycosylation reaction the material was cleaved from the resin. Finally, Wang resin was chosen as the most convenient resin and additional optimization will be studied. As permanent protecting group benzoyl (Bz) group was chosen while fluorenylmethyloxycarbonyl (Fmoc) group was decided as temporal protecting group at position 3. Some previous studies highlighted the risk of migration after deprotection, but our method based on triethylamine treatment showed total deprotection of the Fmoc group and non-migration was observed by NMR technique.
In the modeling and docking experiments that we performed, we could observe that even if long saccharides are biologically important for the stimulation of dendritic cells, at binding site no more than four or five saccharides can be placed. Thus, biological effect might be due to structural and/or valency reasons. Taking into account those observations, we focus on getting short length saccharides and we obtained 4, 5 and 6 mer 1,3-β-glucan.
Further, binding assay based on microarray technique was optimized and ready to be used with our hit compounds.
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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 researcher has obtained some of the candidates of the study, but researcher needs to progress more and synthesize novel compounds. There have been some complications due to the difficulty to deal with these compounds but it is believed that novel candidates might be soon synthesized for biological assays.
The researcher will keep synthesizing 1,3-β-glucan candidates as Dectin-1 ligands. Once the library is obtained, compounds will be evaluated by microarray assay. Further effect of multivalency will be also studied as a novelty of the project. Dr. Lepenies (Max Planck Institute, Germany) provided Dectin1-Fc fusion protein that is the most adequate system to be applied to microarray technique.
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| Strategy for Future Research Activity |
- Synthesis of linear and branched 1,3-1,6-β-glucan.
- Synthesis of multivalency 1,3-β-glucan.
- Microarray assay of obtained compounds with Dectin-1-Fc fusion protein.
- Design and evaluation on a novel glycan array platform based on “double glycoblotting glycan array”.
- Dectin-1 binding of hit candidates will be also evaluated by STD NMR in collaboration with Prof. Jimenez-Barbero (CSIC, Spain).
- Biological assays in vitro and in vivo to determine the availability of short β-glucan as adjuvant.
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| Expenditure Plans for the Next FY Research Funding |
Project budget was saving fin case some unforeseen can happen next year. Further, next year biological assays will be performed, so some of the budget was saved as obtaining biological reagents and assays is supposed to be costly.
Payment of chemical and biological reagents. Payment of material and research facility. Payment of sending compounds to collaborators.
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