title: An optimized ratiometric fluorescent probe for sensing human UDP-glucuronosyltransferase 1A1 and its biological applications.
abstract: This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated.
authors.affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Lv
authors.affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China.
authors.forename: Guang-Bo
authors.initials: GB
authors.lastname: Ge
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Feng
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Troberg
authors.affiliation: College of Life Science, Jilin University, Changchun, China.
authors.forename: Liang-Hai
authors.initials: LH
authors.lastname: Hu
authors.affiliation: Dalian Medical University, Dalian 116044, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Hou
authors.affiliation: Dalian Medical University, Dalian 116044, China.
authors.forename: Hai-Ling
authors.initials: HL
authors.lastname: Cheng
authors.affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Zhao-Ming
authors.initials: ZM
authors.lastname: Liu
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
authors.forename: Moshe
authors.initials: M
authors.lastname: Finel
authors.affiliation: State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China. Electronic address: jncui@dlut.edu.cn.
authors.forename: Jing-Nan
authors.initials: JN
authors.lastname: Cui
authors.affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: yling@dicp.ac.cn.
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology*; D053644:Naphthalimides / Q000737:chemistry* / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods; D000099148:UGT1A1 Enzyme
chemical_list: D005456:Fluorescent Dyes; D014453:Glucuronosyltransferase; D053644:Naphthalimides; D000099148:UGT1A1 Enzyme
keywords: Biological applications; Human UDP-glucuronosyltransferase 1A1; Ratiometric fluorescent probe; Real activity