药学学报, 2017, 52(1): 58-65
引用本文:
丁乐乐, 田镇豪, 侯洁, 翁仔淼, 崔京南, 杨凌, 葛广波. 基于BODIPY母核的羧酸酯酶1特异性荧光探针底物的设计研发[J]. 药学学报, 2017, 52(1): 58-65.
DING Le-le, TIAN Zhen-hao, HOU Jie, WENG Zi-miao, CUI Jing-nan, YANG Ling, GE Guang-bo. Design and development of fluorescent probe substrates for carboxylesterase 1 using BODIPY as the basic fluorophore[J]. Acta Pharmaceutica Sinica, 2017, 52(1): 58-65.

基于BODIPY母核的羧酸酯酶1特异性荧光探针底物的设计研发
丁乐乐1,2, 田镇豪1, 侯洁1,3, 翁仔淼3, 崔京南1, 杨凌2, 葛广波2
1. 大连理工大学精细化工国家重点实验室, 辽宁 大连 116023;
2. 中国科学院大连化学物理研究所, 辽宁 大连 116023;
3. 大连医科大学, 辽宁 大连 116011
摘要:
羧酸酯酶1(carboxylesterase 1,CE1)是哺乳动物体内分布的一种重要的丝氨酸水解酶,广泛参与多种内外源性酯类化合物(包括胆固醇酯等内源物,以及酯类药物及杀虫剂等外源物)的水解代谢。本研究基于人羧酸酯酶1(hCE1)偏好底物的结构特征,选取8-羧酸-BODIPY为荧光母核,设计合成了4种BODIPY-8-羧酸酯衍生物,进而通过单酶筛选和酶抑制实验考察了hCE1催化4种酯类底物的特异性。研究发现,BODIPY-8-羧酸酯的醇基部分越小,hCE1对底物的选择性越高,BODIPY-8-羧酸甲酯(BCM)和BODIPY-8-羧酸乙酯(BCE)均可以作为hCE1的特异性荧光探针底物。在此基础上,选择了水解速率更快的BCM为hCE1探针底物,进一步考察了BCM在人肝微粒体(HLM)和hCE1单酶中的水解动力学,并借助该底物开展了hCE1抑制剂的高效表征研究。研究发现BCM在HLM和hCE1中的酶动力学行为及Km值非常接近,表明hCE1是HLM中参与BCM水解的主要代谢酶。此外,抑制剂表征实验表明BCM可用于hCE1抑制剂的高效筛选与评价,且可用HLM代替hCE1单酶进行酶抑制剂的筛选与评价。
关键词:    羧酸酯酶1      BODIPY母核      荧光探针      特异性      抑制剂筛选     
Design and development of fluorescent probe substrates for carboxylesterase 1 using BODIPY as the basic fluorophore
DING Le-le1,2, TIAN Zhen-hao1, HOU Jie1,3, WENG Zi-miao3, CUI Jing-nan1, YANG Ling2, GE Guang-bo2
1. State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116023, China;
2. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China;
3. Dalian Medical University, Dalian 116011, China
Abstract:
Carboxylesterase 1 (CE1) is an important serine hydrolase in mammals, which involved in the hydrolysis of a variety of compounds (endogenous substrates like cholesterol and xenobiotic compounds like ester-contain drugs and pesticides). This study aimed to design and develop the fluorescent probe substrates for human carboxylesterase 1 (hCE1), on the basis of the structural features of hCE1 preferred substrates. Four carboxylic esters deriving from BODIPY-8-carboxylic acid were designed and synthesized. After then, reaction phenotyping assays and chemical inhibition assays were used to evaluate the selectivity of these four ester derivatives towards hCE1. Our results clearly demonstrated that the substrate specificity of these ester substrates towards hCE1 would be improved with the decrease of the alcohol group on BODIPY-8-carboxylesters, while BODIPY-8-carboxylesters with small alcohol groups including methyl (BCM) and ethyl (BCE) esters could serve as the ideal probe substrates for hCE1. Given that BCM exhibit rapid hydrolytic rate in hCE1, we further investigate the enzymatic kinetics of this fluorescent probe substrate in both human liver microsomes (HLM) and recombinant hCE1, as well as to explore its potential application in high-throughput screening of hCE1 inhibitors by using HLM as enzyme source. The results showed that the kinetic behaviors and the affinity of BCM in HLM is much closed to those in recombinant hCE1, implying that hCE1 played the key roles in BCM hydrolysis in HLM. Furthermore, the inhibition study demonstrated that BCM could be used for rapid screening and characterization of hCE1 inhibitors, by using HLM to replace recombinant hCE1 as enzyme source.
Key words:    carboxylesterase 1    boron-dipyrromethene    fluorescent probe    specificity    inhibitors screening   
收稿日期: 2016-10-17
DOI: 10.16438/j.0513-4870.2016-1004
基金项目: 国家自然科学基金资助项目(81302793,81473181,21572029);精细化工国家重点实验室资助项目(KF1504,KF1408).
通讯作者: 葛广波,Tel/Fax:86-411-84676961,E-mail:geguangbo@dicp.ac.cn
Email: geguangbo@dicp.ac.cn
相关功能
PDF(928KB) Free
打印本文
0
作者相关文章
丁乐乐  在本刊中的所有文章
田镇豪  在本刊中的所有文章
侯洁  在本刊中的所有文章
翁仔淼  在本刊中的所有文章
崔京南  在本刊中的所有文章
杨凌  在本刊中的所有文章
葛广波  在本刊中的所有文章

参考文献:
[1] Teruko I, Masakiyo H. Prodrug approach using carboxylesterases activity:catalytic properties and gene regulation of carboxylesterase in mammalian tissue[J]. J Pestic Sci, 2010, 35:229-239.
[2] laizure SC, herring V, witbrodt K, et al. The role of human carboxylesterases in drug metabolism:have we overlooked their importance[J]. Pharmacotherapy, 2013, 33:210-222.
[3] Homes RS, Wright MW, Laulede SJF, et al. Recommended nomenclature for five mammalian carboxylesterase gene families:human, mouse, and rat genes and proteins[J]. Mamm Genome, 2010, 21:427-441.
[4] Imai T, Taketani M, Shii M, et al. Substrate specificity of carboxylesterase isozymes and their contribution to hydrolase activity in human liver and small intestine[J]. Drug Metab Dispos, 2006, 34:1734-1741.
[5] Ross MK, Crow JA. Human carboxylesterases and their role in xenobiotic and endobiotic metabolism[J]. J Biochem Mol Toxicol, 2007, 21:187-196.
[6] Zhang W, Xu G, McLeod HL. Comprehensive evaluation of carboxylesterase-2 expression in normal human tissues using tissue array analysis[J]. Appl Immunohistochem Mol Morphol, 2002, 10:374-380.
[7] Hosokawa M. Structure and catalytic properties of carboxylesterase isozymes involved in metabolic activation of prodrugs[J]. Molecules, 2008, 13:412-431.
[8] Wang DD, Jin Q, Zou LW, et al. A bioluminescent sensor for highly selective and sensitive detection of human carboxylesterase 1 in complex biological samples[J]. Chem Commun, 2016, 52:3183-3186.
[9] Dominguez E, Galmozzi A, Chang JW, et al. Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes[J]. Nat Chem Biol, 2014, 10:113-121.
[10] Marrades MP, González-Muniesa P, Martínez JA, et al. A dysregulation in CES1, APOE and other lipid metabolismrelated genes is associated to cardiovascular risk factors linked to obesity[J]. Obes Facts, 2010, 3:312-318.
[11] Satoh T, Hosokawa M. The mammalian carboxylesterases:from molecules to functions[J]. Annu Rev Pharmacol Toxicol, 1998, 38:257-288.
[12] Zhu HJ, Wang XW, Gawronski BE, et al. Carboxylesterase 1 as a determinant of clopidogrel metabolism and activation[J]. J Pharmacol Exp Ther, 2013, 334:665-673.
[13] Qian XK, Wang P, Xia YL, et al. A highly selective fluorescent probe for sensing activities of catechol-O-methyltransferase in complex biological samples[J]. Sens Actuators B Chem, 2016, 231:615-623.
[14] Jing Q, Feng L, Wang DD, et al. A two-photon ratiometric fluorescent probe for imaging carboxylesterase 2 in living cells and tissues[J]. ACS Appl Mater Interfaces, 2015, 7:28474-28481.
[15] Dai ZR, Ge GB, Feng L, et al. A highly selective ratiometric two-photon fluorescent probe for human cytochrome P4501A[J]. J Am Chem Soc, 2015, 137:14488-14495.
[16] Lv X, Ge GB, Feng L, et al. An optimized ratiometric fluorescent probe for sensing human UDP-glucuronosyltransferase 1A1 and its biological applications[J]. Biosens Bioelectron, 2015, 72:261-267.
[17] Liu ZM, Feng L, Hou J, et al. A ratiometric fluorescent sensor for highly selective detection of human carboxylesterase 2 and its application in living cells[J]. Sens Actuators B Chem, 2014, 205:151-157.
[18] Jin Q, Feng L, Wang DD, et al. A highly selective nearinfrared fluorescent probe for carboxylesterase 2 and its bioimaging applications in living cells and animals[J]. Biosens Bioelectron, 2016, 83:193-199.
[19] Feng L, Liu ZM, Xu L, et al. A highly selective longwavelength fluorescent probe for the detection of human carboxylesterase 2 and its biomedical applications[J]. Chem Commun, 2014, 50:14519-14522.
[20] Loudet A, Burgess K. BODIPY dyes and their derivatives:syntheses and spectroscopic properties[J]. ChemRev, 2007, 107:4891-4932.
[21] Jiao LJ, Wu YY, Ding Y, et al. Conformationally restricted Aza-Dipyrromethene Boron Difluorides (Aza-BODIPYs) with high fluorescent quantum yields[J]. Chem-Asian J, 2014, 9:805-810.
[22] Yang XD, Zhang XF, Lu XL et al. Red fluorescent monobenzo-BODIPY dyes:solvent effects on spectra and efficient fluorescence quenching by quinones and phenols[J]. J Photochem Photobiol A Chem, 2015, 297:39-44.
[23] Kim SW, Kim HJ, Choi YD, et al. A new strategy for fluorogenic esterase probes displaying low levels of nonspecific hydrolysis[J]. Chem-Eur J, 2015, 21:9645-9649.
[24] Yang L, Ge GB, Zou LW, et al. the inhibitory effect of ursolic acid inhibitors in neutral cholesterol ester hydrolase and its application:CN, 201610866559.1[P]. 2016-9-30.
[25] Aleksandra J, Lauren EJ, Kamilla M, et al. SERS-based monitoring of the intracellular pH in endothelial cells:the influence of the extracellular environment and tumour necrosis factor-α[J]. Analyst, 2014, 140:2321-2329.
[26] Feng L, Liu ZM, Hou J, et al. A highly selective fluorescent ESIPT probe for the detection of human carboxylesterase 2 and its biological applications[J]. Biosens Bioelectron, 2015, 65:9-15.
[27] Liu ZM, Feng L, Ge GB, et al. A highly selective ratiometric fluorescent probe for in vitro monitoring and cellular imaging of human carboxylesterase 1[J]. Biosens Bioelectron, 2014, 57:30-35.
[28] Ge GB, Ning J, Hu LH, et al. A highly selective probe for human cytochrome P4503A4:isoform selectivity, kinetic characterization and its applications[J]. Chem Commun, 2013, 49:9779-9781.
[29] Wang DD, Jin Q, Hou J, et al. Highly sensitive and selective detection of human carboxylesterase 1 activity by liquid chromatography with fluorescence detection[J]. J Chromatogr B, 2016, 1008:212-218.