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

2016 Fiscal Year Annual Research Report

• Project/Area Number: 16F16412
• Research Institution: The University of Tokyo
• Principal Investigator: 菅 裕明 東京大学, 大学院理学系研究科(理学部), 教授 (00361668)
• Co-Investigator(Kenkyū-buntansha): YIN YI-ZHEN 東京大学, 大学院理学系研究科(理学部), 外国人特別研究員
• Project Period (FY): 2016-10-07 – 2019-03-31
• Keywords: ホウ素中性子

Outline of Annual Research Achievements

The aim of this research is to incorporate non-natural boronated amino acids into cyclic peptides so as to discover tumor selective boronated cyclic peptides as boron neutron capture therapy (BNCT) agents by RaPID (random nonstandard peptides integrated discovery) system. In this study, L-boronophenylalanine (L-BPA) and L-carboranylalanine (L-CBA) were chosen because that L-BPA has a similar structure to tyrosine and L-CBA has been shown to be able to replace phenylalanine in biological systems. Indeed, we found that both L-BPA and L-CBA could be efficiently charged onto tRNAs by flexizyme (eFx) technology. Subsequently, it was found that L-BPA could be translated not only at initiation position but also at elongation position by flexible in vitro translation (FIT) system as it was expected. L-CBA could initiate translation although the translation was inefficient probably because of the poor formylation of L-CBA by methionyl-tRNA formyltransferase, indicating that the translation initiation apparatus accepts L-CBA and ribosome can accommodate L-CBA. As the preacetylation of amino acid dramatically increases the expression level in general, ClAc-L-CBA was synthesized and demonstrated to be an efficient initiator that can be used for expression of carborane integrated cyclic peptides by FIT system. The selections provided four L-BPA containing cyclic peptides and eight L-CBA containing peptides which showed strong binding affinity against EGFR with the equilibrium dissociation constant (KD) at low nanomolar level based on the surface plasmon resonance (SPR) measurement.

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

To further confirm the specificity and examine whether the L-BPA and L-CBA integrating peptides could bind to or be internalized into EGFR overexpressed cells, the selected twelve boronated cyclic peptides were successfully synthesized using an automated peptide synthesizer, labeled by fluorescein isothiocyanate (FITC) and purified by HPLC. The further evaluation is now being carried out in Prof. Takigawa’s lab from Kawasaki Medical School. 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 L-CBA were also synthesized. The synthesis of GPNMB or c-Met binding peptides conjugating with multiple L-CBA is also now in progress.

Strategy for Future Research Activity

To evaluate whether the L-BPA or L-CBA integrated peptides could accumulate in cells with sufficient amounts, the boron concentrations in cells including EGFR, GPNMB or c-Met overexpressed 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. It should be mentioned that cooperation with Prof. Junichi Hiratsuka from Kawasaki Medical School and Prof. Koji Ono from Kyoto University will be conducted to carry out this work.