Quantifying protein-drug lifetimes in human cells by 19F NMR spectroscopy.

IF 1.3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Wenkai Zhu, Fatema Bhinderwala, Sarah Rambo, Angela M Gronenborn
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引用次数: 0

Abstract

The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/koff) of a protein-drug complex in human cells by 19F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D 19F-19F exchange spectra were obtained in cells. Essentially identical koff values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by 19F NMR spectroscopy.

用19F核磁共振光谱定量测定人体细胞中蛋白质-药物寿命。
细胞环境是一个复杂而拥挤的空间,细胞器,室室和大量分子参与复杂的通信网络,调节二元蛋白质-配体/蛋白质相互作用。因此,人们越来越认识到,蛋白质与药物结合的评估应该在原生细胞环境中进行。在这里,我们提出了一项概念验证研究,我们使用氟化亲环蛋白a (CypA)结合环孢素a (CsA),通过19F核磁共振光谱测量了人类细胞中蛋白质-药物复合物的寿命(1/koff)。利用CypA中附着在Phe60对位的三氟甲基的特殊检测灵敏度,在细胞中获得了高质量的2D 19F-19F交换光谱。在细胞和体外观察到的koff值基本相同,这表明细胞环境对tfmF60 CypA/CsA复合物寿命的总体影响很小。使用类似的方法来定量天然细胞环境中的蛋白质药物寿命,为通过19F核磁共振波谱有效筛选人类细胞中的药物文库铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Biomolecular NMR
Journal of Biomolecular NMR 生物-光谱学
CiteScore
6.00
自引率
3.70%
发文量
19
审稿时长
6-12 weeks
期刊介绍: The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include: Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR. New NMR techniques for studies of biological macromolecules. Novel approaches to computer-aided automated analysis of multidimensional NMR spectra. Computational methods for the structural interpretation of NMR data, including structure refinement. Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals. New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
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