Dai Zi-Ru, Feng Lei, Jin Qiang, Cheng Hailing, Li Yan, Ning Jing, Yu Yang, Ge Guang-Bo, Cui Jing-Nan, Yang Ling
Dalian Institute of Chemical Physics , Chinese Academy of Sciences , Dalian , China . Email:
Graduate School of Chinese Academy of Sciences , Beijing , China.
Chem Sci. 2017 Apr 1;8(4):2795-2803. doi: 10.1039/c6sc03970g. Epub 2016 Dec 19.
The development of isoform-specific probe(s) for a target enzyme with multiple homologs is always challenging. Herein, a practical strategy was used to design and develop an isoform-specific probe for CYP1A1, a key cytochrome P450 isoenzyme involved in xenobiotic metabolism and bioactivation. On the basis of the subtle differences in 3D structure and substrate preference between CYP1A1 and its homolog CYP1A2, we proposed that it was possible to design a CYP1A1-specific probe local modification of the reaction site on known CYP1A substrates. To validate this hypothesis, 4-hydroxy-1,8-naphthalimide () was selected as the basic fluorophore due to its excellent optical properties, while a series of -alkylated derivatives were prepared to evaluate their specificity towards CYP1A1. Our results revealed that the introduction of a chloroethyl to could get the best isoform selectivity towards CYP1A1 over other CYPs including CYP1A2. The newly developed probe exhibited excellent specificity, high sensitivity, and a ratiometric fluorescence response following CYP1A1-catalyzed -dechloroethylation. was successfully used to real-time monitor the activity of CYP1A1 in complex biological samples and to rapidly screen CYP1A1 modulators in living systems. could also be used for two-photon imaging of intracellular CYP1A1 in living cells and tissues with high ratiometric imaging resolution and deep tissue penetration. All these findings demonstrated that local modification of non-specific substrates was a practical strategy to develop an isoform-specific probe for a target isoenzyme, while could serve as a specific imaging tool to explore the biological functions of CYP1A1 in complex biological systems.
为具有多个同源物的目标酶开发亚型特异性探针一直具有挑战性。在此，我们采用了一种实用策略来设计和开发针对CYP1A1的亚型特异性探针，CYP1A1是参与外源性物质代谢和生物激活的关键细胞色素P450同工酶。基于CYP1A1与其同源物CYP1A2在三维结构和底物偏好上的细微差异，我们提出可以通过对已知CYP1A底物的反应位点进行局部修饰来设计CYP1A1特异性探针。为了验证这一假设，由于其优异的光学性质，选择了4-羟基-1,8-萘二甲酰亚胺作为基本荧光团，同时制备了一系列烷基化衍生物以评估它们对CYP1A1的特异性。我们的结果表明，在4-羟基-1,8-萘二甲酰亚胺上引入氯乙基可以获得对CYP1A1相对于包括CYP1A2在内的其他细胞色素P450的最佳亚型选择性。新开发的探针在CYP1A1催化的脱氯乙基化反应后表现出优异的特异性、高灵敏度和比率荧光响应。该探针成功用于实时监测复杂生物样品中CYP1A1的活性，并在活体系统中快速筛选CYP1A1调节剂。它还可用于活细胞和组织中细胞内CYP1A1的双光子成像，具有高比率成像分辨率和深层组织穿透能力。所有这些发现表明，对非特异性底物进行局部修饰是开发针对目标同工酶的亚型特异性探针的实用策略，而该探针可作为一种特异性成像工具来探索CYP1A1在复杂生物系统中的生物学功能。
