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Wei J.,Tsinghua University | Wei J.,Key Laboratory of Metabolomics at Shenzhen | Jin F.,Tsinghua University | Jin F.,Neptunus Pharmaceutical Technology Center | And 7 more authors.

Human serum albumin (HSA) has been developed as a model protein to study drug-protein interaction. In the present work, the interaction between our synthesized flavonoid derivative 3d (possessing potent antitumor activity against HepG2 cells) and HSA was investigated using fluorescence spectroscopy, circular dichroism spectroscopy, UV-vis spectroscopy and molecular modeling approach. Fluorescence spectroscopy showed that the fluorescence of HSA can be quenched remarkably by 3d under physiological condition with a slight shift of maximum fluorescence emission bands from 360 nm to 363 nm. Calculated results from Stern-Volmer equation and modified Stern-Volmer equation indicated that the fluorescence was quenched by static quenching processing with association constant 5.26±0.04×104 L mol-1 at 298 K. After comprehensive consideration of the free energy change ΔG, enthalpy change ΔH and entropy change ΔS, electrostatic interactions were confirmed as the main factor that participate in stabilizing the 3d-HSA complex. Both dichroism spectroscopy and UV-vis spectroscopy indicated conformational change of HSA after binding to 3d. Moreover, the structure of HSA was loosened and the percentage of α-helix decreased with increasing concentration of 3d. Molecular modeling results demonstrated that 3d could bind to HSA well into subdomain IIA, which is related to its capability of deposition and delivery. Three cation-π interactions and three hydrogen bonds occurred between 3d and amino acid residuals ARG218, ARG222 and LYS199. In conclusion, flavonoid derivative 3d can bind to HSA with noncovalent bond in a relatively stable way, so it can be delivered by HSA in a circulatory system. © 2014 Elsevier B.V. Source

Xie W.,Tsinghua University | Gao D.,Tsinghua University | Gao D.,Key Laboratory of Metabolomics at Shenzhen | Jin F.,Neptunus Pharmaceutical Technology Center | And 3 more authors.
Analytical Chemistry

Single-cell trapping and high-throughput mass spectrometry analysis remain challenging now. Current technologies for single-cell analysis have several limitations, such as throughput, space resolution, and multicomponent analysis. In this study, we demonstrate, for the first time, the combination of microfluidic chip and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput and automatic single-cell phospholipids analysis. A microwell-array-based microfluidic chip was designed and fabricated for cell array formation on an indium tin oxide (ITO)-coated glass slide. Mass spectrometry imaging measurement with 25 μm pixel size was performed with a MALDI ion source. Eight phospholipids in a single A549 cell were detected, and their structures were further identified by MS/MS spectra. Selected ion images were generated with a bin width of Δm/z ± 0.005. The selected ion images and optical images of the cell array showed excellent correlation, and mass spectrometry information on phospholipids from 1-3 cells was extracted automatically by selecting pixels with the same fixed interval between microwells on the chip. The measurement and data extraction could be processed in several minutes to achieve a high-throughput analysis. Through the optimization of different microwell sizes and different matrices, this method showed potential for the analysis of other metabolites or metabolic changes at the single-cell level. © 2015 American Chemical Society. Source

Gao D.,Tsinghua University | Gao D.,Key Laboratory of Metabolomics at Shenzhen | Jin F.,Tsinghua University | Jin F.,Neptunus Pharmaceutical Technology Center | And 3 more authors.

A novel flavonid derivate, 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl) phenyl)-3-(4-chlorophenyl)urea (3d) synthesized in our lab possesses potent antitumor activity against HepG2 cells. Our previous studies on pharmacological mechanism of 3d mostly focused on cell and gene levels, little is about its metabolomics study. Herein, an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of 3d on HepG2 cells and its action mechanism. Q-TOF MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. Comparing 3d-treated HepG2 cells with vehicle control (dimethyl sulfoxide), 32 distinct metabolites involved in glutathione metabolism, glycerophospholipid metabolism, cysteine and methionine metabolism, fatty acid metabolism, and phenylalanine metabolism. The reduced level of glutathione (GSH) and decreased ratio of reduced/oxidized glutathione (GSH/GSSG) in 3d-treated cells indicated the increased oxidative stress after 3d treatment. The significant decrease of phosphatidylcholine (PC) levels and increase of lysophosphatidylcholine (LPC) levels suggested alterations in lipid composition which were causally related to decline in mitochondrial function. Depletion of carnitine and increase of long chain carnitines and fatty acids reflected decline in fatty acid metabolism. The further biological experiments including ROS and MMP measurements confirmed the above probabilities presumed from metabolomic results. Our findings suggested that 3d caused the perturbation of multiple cellular pathways. The increased oxidative stress and the resulting mitochondrial dysfunction resulted in the antiproliferative effect of 3d. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of new compounds that distinct from traditional biological studies. © 2013 Published by Elsevier B.V. Source

Jin F.,Tsinghua University | Jin F.,Neptunus Pharmaceutical Technology Center | Gao D.,Tsinghua University | Wu Q.,Tsinghua University | And 5 more authors.
Bioorganic and Medicinal Chemistry

VEGFR, ERK and Abl had been respectively identified as good drug targets, and their crosstalk also had been well elaborated. Multitarget drugs were more advantageous for cancer treatment, however, no inhibitors simultaneously acting on the three proteins were developed due to their structural diversities. Herein, N-(4-((2-(2-(naphthaen-1-yl)acetamido)ethyl)carbamoyl)piperidin-4-yl)-6- (trifluoromethyl)nicotinamide (NEPT, 6a) was discovered as an active scaffold against VEGFR-2, ERK-2 and Abl-1 kinases through the combination of support vector machine, similarity searching and molecular docking. NEPT and its derivatives were synthesized by convenient routine, their in vitro anti-proliferative abilities against human liver cancer cell line HepG2 were preliminarily evaluated. A representative compound 6b showed an IC50 value of 11.3 μM and induced significant HepG2 cells apoptosis. Besides, these compounds displayed better anti-proliferative abilities against K562 cells (a cell line with typical hyperactivity of the above multikinases), for example compound 6b exhibited an IC50 value of 4.5 μM. Based on hepatotoxicity case reports of Abl inhibitors, cytotoxicity of synthetic compounds against normal liver cell lines (QSG7701 and HL7702) was studied, 6b had a similar toxic effect with positive control imatinib, and most compounds showed less than 35% inhibition activities at 100 μM. Molecular docking study disclosed interactions of 6b with VEGFR-2, ERK-2 and Abl-1 kinases, respectively. Our data suggested the biological activities of 6b may derived from collaborative effects of VEGFR-2, ERK-2 and Abl-1 inhibition. © 2013 Elsevier Ltd. All rights reserved. Source

Gao D.,Tsinghua University | Gao D.,Key Laboratory of Metabolomics at Shenzhen | Chen X.,Tsinghua University | Chen X.,Key Laboratory of Metabolomics at Shenzhen | And 8 more authors.
Journal of the American Society for Mass Spectrometry

The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an 18O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the 18O labeled curcumin was successfully synthesized. The non-labeled and 18O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp.; Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites. © 2015 American Society for Mass Spectrometry. Source

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