2023 Fiscal Year Research-status Report
疾患原因タンパク質を分解するUボディによる新規創薬戦略
| Project/Area Number |
23KF0197
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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) |
PHILIPPE GREGOIRE 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) |
2023-11-15 – 2026-03-31
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| Keywords | Ubiquitin / cancer therapy / proteasomal degradation |
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| Outline of Annual Research Achievements |
- The ability to ubiquitinate a protein using Ubodies (modified ubiquitin) was confirmed.
- Targeting MDM2, an inhibitor of tumor suppressor p53, often requires a helical conformation that mimics the structure of the p53 binding site. New types of selection were developed to improve the helical propensity of the modified loop in Ubodies:
-A) Fully randomized sequence of 8 to 15 amino acid (control)
-B) Randomized sequence (8-15 amino acids) with helix inducers (3-5) on both sides (sequence of amino acids that will help attain the desired structure)
-C) Grafted a sequence known to bind to the target (pDI) and optimized the flanking region (1 to 5 amino acids on both sides) using AlphaFold. The amino acids of the flanking regions are randomized for the selection.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The first selection ((B) helix inducers + randomized sequence) did not show high recovery rate (ratio of protein binding to the beads / total mRNA in the selection). After sequencing, 20 proteins were recombinantly expressed but none of them showed binding to the protein target (MDM2). After optimization, the recovery rate was improved by 100 times and the selection strategy was diversified (as explained in the summary of achievements) to increase the chances of finding binding candidates. A competition selection between (A) and (B) was also performed to attempt to scale binding affinity against ease of translation (D). A delivery method using lipid extracted from cells and manufactured into liposomes is being developed in parallel and will be used once a MDM2 binder Ubody is found.
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| Strategy for Future Research Activity |
The four selection schemes (A-D) will be sequenced, and ~5 binders / selection will be selected for recombinant expression. After binders are found, delivery into cancer cells will be performed using lipofectamine and the reconstituted liposomes. The impact on normal and cancer cell growth will be assessed, and the mechanism of action carefully monitored to conclude on whether the activity comes from protein-protein interaction inhibition (activation of p53 lead to an increase in MDM2 levels due to a negative feedback loop), and/ or degradation of MDM2 by the proteasome (level of MDM2 stay constant or decrease during the assay). Application to other targets will then be performed.
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