【作者】 戴子茹； 葛广波； 冯磊； 金强； 崔京南； 杨凌；

【Author】 Zi-Ru Dai;Guang-Bo Ge;Lei Feng;Qiang Jin;Jing-Nan Cui;Ling Yang;Dalian Institute of Chemical Physics,Chinese Academy of Sciences;State Key Laboratory of Fine Chemicals,Dalian University of Technology;

【机构】 中国科学院大连化学物理研究所； 大连理工大学精细化工国家重点实验室；

【摘要】 CYP1A1是重要的I相代谢酶,主要在人肝外组织中表达,例如人肺、皮肤、小肠中表达。CYP1A1参与多种环境毒素和内源性底物的代谢,并在多种前致癌物被激活成具有遗传毒性中间体或最终致癌物的过程中起到了重要作用,例如在一定程度上激活咖啡因诱使肝硬化的发生。除此之外,CYP1A1的分布存在很大的个体差异,CYP1A1酶的表达受遗传、年龄、疾病、性别、环境和共服药物等多种因素的影响。因此,开展CYP1A1酶活的个体差异研究对于临床个性化安全用药有着重要意义。我们从CYP1A1的活性空腔结构及其偏好底物结构特征入手,以1,8-萘酰亚胺荧光分子为母体,通过预留代谢位点氯乙氧基(发生脱氯乙基化反应)并预留若干化学易修饰基团(提高底物与酶的选择性和灵敏度)的策略,设计合成了一种高选择性双光子比率型CYP1A1荧光探针分子。该分子与其去氯乙基化产物显示了不同的光学属性,NBCeN及其产物NBHN的最大激发波长分别为372和450 nm,最大发射波长分布为452和562 nm,显示该探针分子反应前后有较大的斯托克斯位移(110 nm),且其定量检测下限为0.025 nmol/mL。体外人肝微粒体(HLM)CYP代谢孵育体系中检测到仅有一个去氯乙基化代谢产物生成。NBCeN在人肝微粒体(HLM),人肺微粒体(HLuM)和CYP1A1单酶体系中动力学参数K_m分别为1.79±0.09,0.36±0.03和0.84±0.07μM,相似的K_m值提示NBCeN在HLM和HLuM去氯乙基化中CYP1A1起了主要作用。同时体外化学抑制实验显示CYP1A1的特异性抑制剂白藜芦醇可强烈抑制NBCeN在HLM的CYP代谢孵育体系中的反应。利用该荧光探针反应,定量测定了12例个体人肝微粒体中的CYP1A1酶的活性,且12例个体人肝微粒体中NBCeN的去氯乙基化速率和乙氧基试卤灵(广泛使用的CYP1A1探针底物)的去乙基化速率高度相关,相关系数为0.99,该结果表明该荧光探针分子具有良好的选择性。此外,该双光子荧光探针还可以用于CYP1A1抑制剂和诱导剂的筛选。而且首次实现了活细胞与活组织层面的CYP1A1酶活性的比率型双光子荧光成像。该探针不仅适用于单酶、细胞及组织中CYP1A1酶活的实时定量检测,还可用于新药研发早期筛选、组织成像及体外诊断等生物医药相关领域。更多还原

【Abstract】 Human cytochrome P450 1A1(CYP1A1) is an important phase I drug-metabolizing enzymes involved in the activation of procarcinogenic compounds to their ultimate carcinogens.In this study,a two-photon ratiometric fluorescent probe NBCeN has been designed and developed for highly selective and sensitive detection of CYP1A1 for the first time.The probe was designed based on catalytic function and substrate preference of CYP1A1,as well as two-photon absorption properties of naphthalimide fluorophore.A series of O-alkylated 4-Hydroxy-1,8-naphthalimide(HN) derivatives were purposely prepared to evaluate their potential as good substrates of CYP1A1 through docking and reaction phenotyping screening.Our results demonstrated that the introduction of a chloroethyl to HN could significantly improve the selectivity of CYP1A1.And NBCeN successfully exhibited a ratiometric fluorescent response at 452 nm and 562 nm and the detection limit was about 0.025 nmol/mL,indicated that the probe with high sensitivity.Only one metabolite was detected when NBHN was incubated with the human liver microsomes along with the NADPH-generating system.The metabolite was identified as N-(4-butyl)-4-hydroxy-l,8-naphthalimide(NBHN) by using LC-MS and NMR techniques.Kinetic characterization demonstrated that NBHN in HLM,HLuM and CYP1A1 enzyme displayed the similar apparent kinetic parameters,which revealed CYP1A1 played an important role in NBCeN human tissue microsome O-dechloroethylation.Furthermore,resveratrol,a specific inhibitor of CYP1A1,strongly inhibited NBCeN O-dechloroethylation in HLM.Meanwhile,in 12 cases of individual human liver microsomes O-dechloroethylation reaction rates of NBCeN and O-deethylation reaction rate of ethoxyresorufin(widely used as a CYP1A1 probe substrate) were highly correlated,the correlation coefficient was 0.99.This probe could also screen CYP1A1 modulators by using tissue preparations as the enzymes sources.Additionally,the two-photon excited fluorescence properties has made NBCeN excellent use for imaging of intracellular CYP1A1 in living cells and tissues,and showed high ratiometric imaging resolution and deep-tissue imaging depth.All these findings suggested that NBCeN hold great potentials as a highly practical two-photon excited ratiometric fluorescent sensor for monitoring CYP1A1 activities in complex biological systems and for exploring the biological functions and medicinal roles of CYP1A1 in living systems.更多还原