| Uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1), an important phase ? drug-metabolizing enzymes in human, is responsible for the metabolism and elimination of the endobiotics toxic metabolite bilirubin and many xenobiotics, and its dysfunction has been proved to be closely related to many diseases, such as hyperbilirubinemia, jaundice, hepatotoxicity, neurotoxicity and personalized medication. Hence, it is of great importance to evaluate and characterize the activity of UGT1A1.Currently, the quantification of UGT1A1 activity is mainly accomplished with the help of endobiotics and xenobiotics substrates, such as bilirubin and drugs. However, available probes for UGT1A1 are quite limited and existing problems of selectivity, sensitivity, stability and practicability. Hence, the real-time assessment of enzyme activity of UGT1A1 in complex biological system has been a challenge in drug metabolism and biological analysis.Fluorescent probes for functional enzymes have attracted considerable attention due to their inherent advantages, such as high sensitivity, high-throughput screening, high resolution and applicability to in situ imaging, which can overcome the drawbacks of traditional probes. However, the fluorescence properties of many fluorophores are often "turned-off' following glucuronidation by UGT1A1, which is the most difficult problem in designing UGT1A1 fluorescent probes. In addition, UGTs are a family of membrane protein, which can only be quantified or imaged in the presence of intact membrane structure and cofactor. Thus, a fluorophore with outstanding optical properties was selected to design and synthesize a series of compounds for this study, aiming to develop a fluorescent probe for highly selective and sensitive detection of human UGT1A1 through selectivity optimization. The main results are as follows:(1) Development of UGT1A1 OFF-ON fluorescent probe. Taking into account the substrate preference of UGT1A1 and its vital capacity for catalyzing O-glucuronidation towards polycyclic phenolic compounds,1,8-naphthalimide was selected as the basic fluorophore due to its outstanding optical properties,3 phenol-1,8-naphthalimide compounds with hydroxy in different position were then designed and synthesized by elongating the ?-conjugate resonance of 1,8-naphthalimide rings. Incubation systems with human liver microsomes (HLM) and recombinant UGT isoforms were applied to evaluate the UGT1A1 selectivity and to explore the potential structure-UGT1A1 selectivity relationship, the results demonstrated that the substitution of phenolic hydroxyl in para position could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, N-butyl-4-(4-hydroxyphenyl)-1,8-naphthalimide (NPHN) exhibited excellent specificity towards human UGT1A1, only metabolized by UGT1A1. NPHN was not a fluorescent compound by itself, but its metabolite (NPHN-O-glucuronide) was with fluorescent properties, emission wavelength at 520 nm and Stokes shift of 150 nm, which makes it to be an OFF-ON fluorescent probe for UGT1A1. Thirdly. NPHN has been successfully applied for the measurement of the UGT1A1 activities in 14 individual HLM, and the results demonstrated that there was a strong correlation with the UGT1A1 activity determined by traditional probes (R2>0.9) and about 10-fold variation between the highest and lowest activity rates. Finally, NPHN was a good probe for human UGT1A1 and enabled imaging of UGT1A1 activity in HepG2 cells. In summary, NPHN was the first high selectivity and high sensitivity OFF-ON fluorescent probe for human UGT1A1.(2) Development of UGT1A1 ratiometric fluorescent probe. Ratiometric fluorescent probes allow the measurement of target(s) by using the ratio of two distant fluorescent signals, thus exhibiting some advantages, including minimization of the external interference and alleviation of the shortcomings of intensity-based probes in quantitative detection. It is thus highly desirable to develop a more accurate and sensitive ratiometric fluorescent probe to measure the real activity of UGT1A1 in human biological samples. Different from the abovementioned modification strategy in elongating the ?-conjugate resonance of 1,8-naphthalimide rings, a series of N-substituted derivatives of 4-hydroxy-1,8-naphthalimide ring were then designed and synthesized, constructing a library of 15 fluorescent compounds. Incubation systems with HLM and recombinant UGT isoforms were applied to evaluate UGT1A1 selectivity and to explore the potential structure-UGT1A1 selectivity relationship and the results demonstrated that the introduction of an acidic group to N-position could significantly improve the selectivity of UGT1A1. N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN) exhibited excellent specificity towards human UGT1A1, and there was more than 26-fold difference in the selectivity between UGT1 Al and other human UGT isoforms. NCHN displayed different fluorescence properties with its metabolite (NCHN-4-O-glucuronide), which makes it to be a ratiometric fluorescent probe. NCHN has been successfully applied for the measurement of the UGT1A1 activities in 14 individual HLM, and the results demonstrated that there was a strong correlation with the UGT1A1 activity determined by traditional probes (R2> 0.9) and about 10-fold variation between the highest and lowest activity rates. In addition, NCHN was a good probe for human UGT1A1 and enabled ratiometric imaging of UGT1A1 activity in HepG2 cells. In summary, NCHN was the first high selectivity and high sensitivity ratiometric fluorescent probe for human UGT1A1, avoiding the problems of multiple metabolism sites, low selectivity and sensitivity of UGT1A1 probes used before.(3) High-throughput screening of UGT1A1 inhibitors. In order to study the relationship of UGT1A1 and ligand and explore the influences of traditional Chinese mediation on UGTs, a fluorescence based high-throughput screening method for the discovery of UGT1A1 inhibitors was developed, using NCHN as the specific fluorescent probe substrate for UGT1A1. Fructus Psoraleae (FP) was selected as the model traditional Chinese herbal, and a liquid chromatography with ultraviolet detection (LC-UV) fingerprint for Fructus Psoraleae AND UGT1A1 inhibition profile was established. The results demonstrated that five major components of FP displayed evident inhibitory effects on UGT1A1, among which bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (Ki) values lower than 1 ?M. while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the Ki values ranging from 1.61 to 10.0 ?M. Meanwhile, the most commonly used nonselective substrate 4-MU was used in parallel for evaluation of UGT1A1 inhibitors in vitro based on LC-UV method, demonstrating that these five major components of FP displayed similar inhibition tendency and different inhibition kinetic behaviors against UGT1A1. The results implied that this method can be applied to high-throughput screening of UGT1A1 inhibitors in complex traditional medicine and predict the drug-drug interaction mediated by UGT1A1, and NCHN and 4-MU may bind with different regions or sub domains of UGT1A1.(4) Primary study of two fluorescent probes in UGT1A1 active sites. In order to explore the binding site of different ligand, the relationship of UGT1A1 and ligand, and to explore whether NCHN and NPHN could be used as a couple for future inhibition-related studies on UGT1A1, enzyme chemical inhibition kinetic and enzyme mutant kinetic study were performed to investigate the UGT1A1 active sites for NCHN and NPHN. These two highly selective UGT1A1 fluorescent probes namely NCHN and NPHN were demonstrated to be typical substrate of UGT1A1 with different active sites through enzyme inhibition kinetics and catalytic kinetics from UGT1A1 mutants. The results suggested that NCHN and NPHN may serve as two different classes of UGT1A1 substrates which may bind with UGT1A1 at two different active sites. Therefore, NCHN and NPHN could be used as a couple for future inhibition-related studies on UGT1A1, avoiding the occurrence of adverse drug-drug interaction mediated by UGT1A1.In conclusion, two different types (ratiometric fluorescent probe and OFF-ON fluorescent probe) high selectivity and sensitivity fluorescent probes for human UGT1A1 were obtained. The newly developed two fluorescent probes have been successfully applied for the measurement of the UGT1A1 activities in different biological samples, the bioimaging of UGT1A1 in human liver cells, as well as rapid screening of UGT1A1 inhibitors from Chinese traditional herbs. In addition, NCHN and NPHN were demonstrated to be two different classes of typical UGT1A1 substrate which bind with UGT1A1 at two different active sites. This study obtained UGT1A1 fluorescent probes used for activity phenotyping by rational design, which laid a solid foundation for the future studies on the phenotyping of UGT1A1 activity with both high throughput and high content as well as.UGT1A1-mediated toxicology study. |
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