2017 Fiscal Year Annual Research Report
中性子捕捉療法を指向したホウ素含有環状ペプチドの開発
| Project/Area Number |
16F16412
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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) |
YIN YI-ZHEN 東京大学, 大学院理学系研究科, 外国人特別研究員
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| Project Period (FY) |
2016-10-07 – 2019-03-31
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| Keywords | BNCT / macrocyclic peptides / RaPID / Bpa / Cba / hEGFR |
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| Outline of Annual Research Achievements |
We synthesized α-N-(2-choloroacetyl)-L-boronophenylalanine cyanomethyl ester (ClAc-Bpa-CME), L-boronophenylalanine cyanomethyl ester (Bpa-CME) and α-N-(2-choloroacetyl)-L-carboranylalanine cyanomethyl ester (ClAc-Cba-CME), which were subsequently charged onto tRNAs by enhanced flexizyme (eFx). Then it was demonstrated that ClAc-Bpa and ClAc-Cba could initiate the peptide synthesis, and Bpa could be arranged in the elongation position. This is the first time to incorporate Cba into peptide chains by ribosome. We next applied ClAc-Bpa, Bpa and ClAc-Cpa into RaPID (random nonstandard peptide integrated discovery) system for screening boronated cyclic peptides against hEGFR, Four peptides from Bpa family and,eight peptides from Cba family exhibited strong binding affinities to hEGFR. Furthermore, the selected twelve peptides bearing Bpa and Cba were labeled with fluorescein isothiocyanate (FITC) and their binding specificity to hEGFR expressing cells were evaluated using fluorescence microscopy. It is found that the HEK293-hEGFR expressing cells were obviously stained by FITC labeled BpaP5, BpaP13, CbaP5, CbaP14 and CbaP16 peptides as compared to invisible staining for the HEK-293 mock cells, suggesting their binding specificity against hEGFR. Next, we also replaced the Bpa or Cba in peptides into L-phenylalanine (F), it was found that the boric acids in Bpa bearing peptides might not be involved in the interaction with hEGFR. Interestingly, carborane in Cba bearing peptides was demonstrated to be responsible for binding to hEGFR.
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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
Since BpaP5, BpaP13, CbaP5, CbaP14 and CbaP16 peptides could definitely bind to hEGFR expressing cells, it will be promising that the Bpa and Cba integrating peptides could also be internalized into the cells. The further evaluation for this part is now being carried out in Prof. Takigawa’s lab from Kawasaki Medical School.
It is anticipated that introducing some non-proteinogenic amino acids can enhance the peptides protease resistance. As it is unknown that introducing Bpa or Cba into peptides could prevent the peptides degradation by protease to some degree, the screening assay for determination of the peptides stability in human serum or plasma will be performed. Since 1,14-cyclic BpaP13 and CbaP5 represent the two strongest binding peptides against hEGFR, serum stability evaluation of these two peptides as well as their mutants peptides 1,14-cyclic BpaP13-1F/3F and CbaP5-1F are now in progress.
Since two peptides that could strongly bind to and be respectively internalized into GPNMB or c-Met overexpressed cells were discovered in our lab, the conjugates between GPNMB or c-Met binding peptides and Cba were also synthesized. It was investigated that post-modification with Cba had little or no effect on the activity of the original peptides. We next tried to add two Cba to the peptides, but it looks that it can reduce half of the binding affinity according to the FACS results compared with original peptides. Further evaluation and optimization are now in progress.
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| Strategy for Future Research Activity |
To evaluate whether the Bpa and Cba integrated peptides could accumulate in cells with sufficient amounts, the boron concentrations in hEGFR overexpressing cells, will be measured by inductively coupled plasma-atomic emission analysis (ICP-AES). Furthermore, biodistribution studies will be performed in tumor-bearing mice, and boronated cyclic peptides will be administered intravenously or intraperitoneally. The boron concentrations in tumors and normal tissues including brain, blood, liver, kidney and muscle will be measured by ICP-AES or prompt γ-ray spectrometer again. Finally, the selected peptides will be evaluated to check the effectiveness by thermal neutron exposure in mice.
If the screened boronated cyclic peptides can exhibit high selectivity and internalization rate, they will be conjugated with DNA intercalators, such as doxorubicin. By this manner, DNA intercalators tend to selectively target tumor under the help of tumor penetrating peptides, and boronated cyclic peptides might be accumulated around DNA due to the contribution of DNA intercalators, substantially reducing the boron amount required for BNCT. Moreover, DNA intercalators having fluorescence may provide a research tool for fluorescent imaging and quantification of peptides uptake and biodistribution in the cells. Different linkers will be tested to retain the binding affinity and internalization activity of boronated cyclic peptides, and to construct firm linkage between peptides and DNA intercalators.
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