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Liu W.,Tsinghua University | Liu W.,Key Laboratory of Metabolomics at Shenzhen | Jin F.,Tsinghua University | Jin F.,Neptunus Pharmaceutical Technology Center | And 7 more authors.
RSC Advances | Year: 2017

A novel aminoquinazolin derivative (named 9d), synthesized in our lab shows potential antitumor activity against A549 lung cancer cell lines. However, previous studies on the pharmacological mechanism of 9d mostly focused on cell and gene levels; the metabolic mechanism remains unknown. In this paper, an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to study the probable antitumor mechanism. Q-TOF MS and MS/MS were used to identify significantly different metabolites. 22 significantly different metabolites were observed between the 9d-treated A549 lung cells group and control group. They were involved in glycerophospholipid metabolism, glutathione metabolism, phenylalanine metabolism, cysteine and methionine metabolism, and aminoacyl-tRNA biosynthesis. The results showed that glutathione level and cell-membrane components phosphatidylcholines were decreased in 9d-treated cells, while their oxidative products, oxidized glutathione and lysophosphatidylcholines, were significantly increased. Further biological investigation showed an apparent accumulation of reactive oxygen species and a decrease in mitochondrial membrane potential. The results indicated that the aminoquinazolin derivative induced oxidative stress-mediated apoptosis. Moreover, phenylalanine metabolism suggested the up-regulation of l-phenylalanine might act as an endogenous drug carrier of 9d to improve cellular antiproliferation ability. The inhibition of aminoacyl-transfer RNA and flow cytometry results indicated that cell cycle progression was blocked in the G1 phase, which was in accordance with the results obtained from some marketed aminoquinazolin derivatives. The above results proposed that 9d could induce oxidative stress and cell cycle arrest, which finally led to cell apoptosis in A549 cells. The study supplies a rapidly and highly active strategy to investigate the antitumor mechanism of drugs, which benefits its further improvement and development. © The Royal Society of Chemistry.


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 | Year: 2013

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.


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.
Talanta | Year: 2014

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.


Shao X.,Tsinghua University | Gao D.,Tsinghua University | Gao D.,Key Laboratory of Metabolomics at Shenzhen | Wang Y.,Tsinghua University | And 5 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2016

Flavopiridol is reported to have potent antitumor effects by inhibition of cyclin-dependent kinases (CDKs). However, most studies of flavopiridol focus on specific genes and kinases, so the antitumor mechanism needs further elucidation at the metabolic level. In the present study, an UPLC/Q-TOF MS metabolomics approach was used to investigate its antiproliferative effects on MCF-7 breast cancer cells. Comparing flavopiridol-treated MCF-7 cells with vehicle control, 21 potential biomarkers involved in five metabolism pathways were identified. Two pathways involving glutathione metabolism and glycerophospholipid metabolism showed that glutathione (GSH) and phosphatidylcholines (PCs) levels were reduced while their oxidized products oxidized glutathione (GSSG) and lysophosphatidylcholines (LysoPCs) were greatly increased. Further investigation showed an apparent accumulation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). Thus, we suggest that oxidative stress was provoked in MCF-7 cells to reduce the GSH and PCs levels and cause mitochondria lesions. Moreover, cell cycle analysis showed that flavopiridol blocked cells at G1 stage, which was consistent with the depletion of spermidine and spermine that are believed to promote cancer progression. Taking these together, we concluded that flavopiridol could induce oxidative stress and cell cycle arrest, which finally lead to cell apoptosis in MCF-7 cells. This study provides a new strategy for studying the antitumor mechanism of flavopiridol, which could be used for its further improvement and application. © 2016 Elsevier B.V.


Yang T.,Tsinghua University | Gao D.,Tsinghua University | Jin F.,Neptunus Pharmaceutical Technology Center | Jiang Y.,Tsinghua University | Liu H.,Tsinghua University
Rapid Communications in Mass Spectrometry | Year: 2016

Rationale: Single-cell analysis is very important in several research fields for the heterogeneity of individual cells, which has been well accepted. However, restricted by the size and low content of a single cell, current studies have encountered challenges in high-throughput, high-space resolution and sensitivity, and multicomponent analysis. A methodology of a surface-printed microdot array chip coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is presented in this study for high-throughput single-cell patterning and phospholipid analysis. Methods: The poly-L-lysine (PLL) used as ink molecule was printed on an oxygen plasma processed indium tin oxide (ITO)-coated glass slide to form a microdot array by micro-contact printing technology. The cell array was then formed on the PLL microarray through electrostatic adsorption force. 9-Aminoacridine (9-AA) matrix was applied on the cell array before it was analyzed by MALDI-TOF MS. MALDI mass spectrometry imaging (MALDI-MSI) was then used for high-throughput, quick measurement, and multicomponent analysis of the cell array. Results: The single-cell capture efficiency of the cell array formed on the PLL microarray was about 40%. Twelve phospholipids were detected at the single-cell level, and the structures were further confirmed by MS/MS. The MALDI-MSI of selected ions showed a conformity with the cell array. The relative signal intensity data of selected ions were extracted from every pixel in the image within several minutes. The heterogeneity between individual cells was revealed from the relative signal intensity of phospholipids in 1–3 cells. Conclusions: Compared to the existing related approaches, high-throughput, quick measurement, and multicomponent single-cell analysis have been realized by our method. Through different ink molecules used for micro-contact printing, the established platform could have the potential to capture and analyze specific cells. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.


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 | Year: 2015

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.


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.
Talanta | Year: 2014

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.


Shao X.,Tsinghua University | Gao D.,Tsinghua University | Gao D.,Key Laboratory of Metabolomics at Shenzhen | Chen Y.,Tsinghua University | And 6 more authors.
Analytica Chimica Acta | Year: 2016

Since most of the central nervous system (CNS) drug candidates show poor permeability across the blood-brain barrier (BBB), development of a reliable platform for permeability assay will greatly accelerate drug discovery. Herein, we constructed a microfluidic BBB model to mimic drug delivery into the brain to induce cytotoxicity at target cells. To reconstitute the in vivo BBB properties, human cerebral microvessel endothelial cells (hCMEC/D3) were dynamically cultured in a membrane-based microchannel. Sunitinib, a model drug, was then delivered into the microchannel and forced to permeate through the BBB model. The permeated amount was directly quantified by an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after on-chip SPE (μSPE) pretreatment. Moreover, the permeated drug was incubated with glioma cells (U251) cultured inside agarose gel in the downstream to investigate drug-induced cytotoxicity. The resultant permeability of sunitinib was highly correlated with literature reported value, and it only required 30 min and 5 μL of sample solution for each permeation experiment. Moreover, after 48 h of treatment, the survival rate of U251 cells cultured in 3D scaffolds was nearly 6% higher than that in 2D, which was in accordance with the previously reported results. These results demonstrate that this platform provides a valid tool for drug permeability and cytotoxicity assays which have great value for the research and development of CNS drugs. © 2016 Elsevier B.V.


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 | Year: 2015

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.


PubMed | Tsinghua University and Neptunus Pharmaceutical Technology Center
Type: | Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences | Year: 2016

Flavopiridol is reported to have potent antitumor effects by inhibition of cyclin-dependent kinases (CDKs). However, most studies of flavopiridol focus on specific genes and kinases, so the antitumor mechanism needs further elucidation at the metabolic level. In the present study, an UPLC/Q-TOF MS metabolomics approach was used to investigate its antiproliferative effects on MCF-7 breast cancer cells. Comparing flavopiridol-treated MCF-7 cells with vehicle control, 21 potential biomarkers involved in five metabolism pathways were identified. Two pathways involving glutathione metabolism and glycerophospholipid metabolism showed that glutathione (GSH) and phosphatidylcholines (PCs) levels were reduced while their oxidized products oxidized glutathione (GSSG) and lysophosphatidylcholines (LysoPCs) were greatly increased. Further investigation showed an apparent accumulation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). Thus, we suggest that oxidative stress was provoked in MCF-7 cells to reduce the GSH and PCs levels and cause mitochondria lesions. Moreover, cell cycle analysis showed that flavopiridol blocked cells at G1 stage, which was consistent with the depletion of spermidine and spermine that are believed to promote cancer progression. Taking these together, we concluded that flavopiridol could induce oxidative stress and cell cycle arrest, which finally lead to cell apoptosis in MCF-7 cells. This study provides a new strategy for studying the antitumor mechanism of flavopiridol, which could be used for its further improvement and application.

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