| Project/Area Number |
22K15643
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 51030:Pathophysiologic neuroscience-related
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2024: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2023: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | alpha synuclein / amyloid fibrils / fibril morphology / amyloidosis / alpha Synuclein / amyloid fibril / polymorphism / Parkinson's disease |
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| Outline of Research at the Start |
①化合物AがαSyn構造スイッチを行い、弱毒化する機序の解明：初年度～2年目我々はすでに化合物AがαSynと結合すること、そして弱毒化することを見出している。どのようにして数分での凝集体構造変換を可能にしているのか、機序を明らかにするとともに、毒性の低い凝集体について、何が弱毒化を規定しているのかについて解析する。
②構造展開や他化合物の取得：2年目～3年目同様の薬効をもつ化合物をスクリーニングし、構造変換効率のより高い薬を導出する。化合物Aは分子量が大きく、BBB通過しないため、構造活性相関を解析し、最小限の構造を残した化合物展開を行う(大阪大学薬学部と連携)
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| Outline of Annual Research Achievements |
1) Expression of recombinant alpha synuclein and amyloid fibril formation. There is a constant production of recombinant alpha synuclein to keep generating amyloid fibrils with them.
2) The visualization of mature amyloid fibrils has been performed using transmission electron microscopy. It has been possible to detect the presence of a reproducible morphology when amyloid fibrils are treated with a variety of compounds similar to the polyphenol compound A.
3) Molecular mechanisms of morphology switching. We have designed a strategy that involves a partial digestion of amyloid fibrils with proteases in order to obtain fragments that are planned to be analyzed by mass spectrometry. This tool is expected to corroborate the chemical reaction.
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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
1. At this point, the method for protein purification has totally been established, including controls of the protein purity and its performance at forming amyloid fibrils.
2. The morphology of the fibrils treated with the compound A and its derivatives is consistent from reaction to reaction, independently of the compound employed. This strongly suggests a common chemical event that takes place in all the reactions evaluated.
3. We have reconsidered the strategy to obtain chemical evidences that corroborate the same chemical reaction is taking place with all the compounds tested. Currently we have adjusted the original plan in order to integrate spectroscopic tools, more specifically mass spectrometry and 1D nuclear magnetic resonance, to provide a general mechanism of action. Eventually, we think the results can be employed to designed an optimized structure that shows an improved performance when reacting with amyloid fibrils.
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| Strategy for Future Research Activity |
Since the current status of the project, after adjusting the chemical strategy, seems to be in track with the originally planned objectives, we are considering to continue with the next stage of the experimental schedule:
1) Complete the chemical characterization of the reaction that takes place between the fibrils and the compounds. Essentially through nuclear magnetic resonance and mass spectrometry. One of the difficulties found was the technical limitation of the size of the switched morphologies to be analyzed by mass spectrometry and NMR. Now we have designed a pretreatment with proteases that more specifically cleave the reacted section of the fibrils and facilitate the spectroscopic analyses.
2) With the spectroscopic evidences, we plan to use the results to design an improved compound with better characteristics that the currently available in our materials. Two options will be explored: 1) similar compounds that show structural similarities; 2) evaluate a chemical synthesis of an optimized compound
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