Real-Time Quantitative Analysis of Drug Deliveries by In-Cell NMR

2018 Fiscal Year Final Research Report

• Project/Area Number: 15K05401
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Physical chemistry
• Research Institution: Himeji Dokkyo University
• Principal Investigator: OKAMURA Emiko 姫路獨協大学, 薬学部, 教授 (00160705)
• Co-Investigator(Kenkyū-buntansha): 安岐 健三 姫路獨協大学, 薬学部, 助手 (50714945)
• Research Collaborator: Tohyama Yumi
• Project Period (FY): 2015-04-01 – 2019-03-31
• Keywords: 超精密計測 / 生物物理 / リアルタイム解析 / In-Cell NMR / 薬物輸送

Outline of Final Research Achievements

Real time in-cell NMR spectroscopy was applied to quantify kinetics of non-endocytic membrane permeation of octaarginine (R8) into living cells. The non-endocytic membrane translocation of 19F-labeled R8 into a human myeloid leukemia cell line was observed at 4 °C with a time resolution of minute-order. The 19F NMR detected the real time translocation of cationic R8: the binding to anionic glycosaminoglycan at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. Our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of membrane-permeable peptides like R8 in cell membranes.
The methods were also applied to analyze spontaneous peptide bond cleavage at natural L-α-Asp and abnormal D-β-Asp isomers in eye lens α-crystallin fragments in real time. Kinetic analysis showed how tough the uncommon D-β-Asp residue was to allow abnormal accumulation.

Free Research Field

生物物理化学

Academic Significance and Societal Importance of the Research Achievements

生きた細胞への物質輸送のNMRリアルタイム定量計測はこれまで類例がなく、薬物の輸送機構を解明する新しいアプローチとして、薬の作用や毒性予測、新薬の探索やデザインに向けて、研究のもつ意義は大きい。
また、白内障やアルツハイマー病関連ペプチドを対象としたペプチド中のアミノ酸の異性化、ペプチド結合切断のNMRリアルタイム計測は、今後、加齢に伴う疾患のメカニズム解明とその制御、予防や創薬に向けて、物理化学に基づく新たな視点からのアプローチとなることが期待される。