Publications by authors named "Jingwu Kang"

50 Publications

Screening inhibitors for blocking UHRF1-methylated DNA interaction with capillary electrophoresis.

J Chromatogr A 2021 Jan 9;1636:461790. Epub 2020 Dec 9.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China; School of physical science and technology, ShanghaiTech University, Haike Road 100, Shanghai, 200120, China. Electronic address:

Epigenetic inheritance in mammals relies in part on propagation of DNA methylation patterns throughout development. UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) is required for maintenance the methylation pattern. It was reported that UHRF1 is overexpressed in a number of cancer types, and its depletion has been established to inhibit growth and invasion of cancer cells. It has been considered as a new therapeutic target for cancer. In the present work, we described a method for screening inhibitors for blocking the formation of UHRF1-methylated DNA (mDNA) complex by using nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM). A recombinant UHRF1 with the SRA domain (residues 408-643), a fluorescently labeled double strand mDNA (12 mer) and a known inhibitor mitoxantrone were employed for proof of concept. The method allows to measure the dissociation constant (K) of the UHRF1-mDNA complex as well as the rate kinetic constant for complex formation (k) and dissociation (k). A small chemical library composed of 60 natural compounds were used to validate the method. Sample pooling strategy was employed to improve the screening throughput. The merit of the method was confirmed by the discovery of two natural products proanthocyanidins and baicalein as the new inhibitors for blocking the formation of UHRF1-mDNA complex. Our work demonstrated that CE represents a straightforward and robust technique for studying UHRF1-mDNA interaction and screening of the inhibitors.
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http://dx.doi.org/10.1016/j.chroma.2020.461790DOI Listing
January 2021

A gold foil covered fused silica capillary tip as a sheathless interface for coupling capillary electrophoresis-mass spectrometry.

J Chromatogr A 2020 Aug 17;1624:461215. Epub 2020 May 17.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China; School of Physical Science and Technology, ShanghaiTech University, Haike Road 100, Shanghai 200120, China. Electronic address:

A method for the preparation of an on-column ESI emitter used as the sheathless interface for coupling capillary electrophoresis (CE) with mass spectrometry (MS) was developed. The emitter was directly fabricated at the outlet end of the separation capillary which was etched with HF solution to a symmetrical tip. The tip was covered with a small piece of gold foil which was fixed by epoxy resin glue for electrical contact. Such a prepared ESI emitter can produce a stable ESI signal over the wide range of flow rate from 50 nL/min to 800 nL/min. The performance of the CE-MS with the sheathless interface was evaluated by using the separation of four alkaloids. It was found that the strong electroosmotic flow produced by the multiple polyelectrolyte coating on the capillary is necessary for maintaining a stable MS signal. Effect of the running buffer composition, concentration and the CE separation voltages on the ESI signal strength were investigated. The absolute detection limits for the alkaloids was determined as fmol level. Moreover, the CE-MS was applied for the analyses of trypsin digestion of cytochrome C and small molecular organic anions. The emitter performed very stable with a lifetime of at least 180 h.
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http://dx.doi.org/10.1016/j.chroma.2020.461215DOI Listing
August 2020

Probing Protein-Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns.

Anal Chem 2020 03 12;92(5):3913-3922. Epub 2020 Feb 12.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.
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http://dx.doi.org/10.1021/acs.analchem.9b05355DOI Listing
March 2020

Separation of twelve posaconazole related stereoisomers by multiple heart-cutting chiral-chiral two-dimensional liquid chromatography.

J Chromatogr A 2020 May 2;1618:460845. Epub 2020 Jan 2.

State key laboratory of Bioorganic Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Lu, Shanghai 200032, China; School of physical science and technology, ShanghaiTech University, Haike Road 100, Shanghai 200120, China. Electronic address:

Posaconazole represents a triazole antifungal agent which is used to treat various fungal infections. It contains four chiral centers leading to 16 stereoisomers. With the convergent synthesis route, only 11 related stereoisomeric impurities may potentially exist in the active pharmaceutical ingredient (API). It is a challenge to separate all the stereoisomers in one run. To address this problem, a multiple heart-cutting chiral-chiral two-dimensional liquid chromatography (2D-LC) method was developed. The multiple heart-cutting 2D-LC separation was implemented on 2D-LC system with three chiral columns with immobilized polysaccharide chiral stationary phases, namely Chiralpak IB, IC and IF3. In the system, the column Chiralpak IB was used as the D separation column and IC and IF3 columns were used parallelly for the D separation. The twelve stereoisomers were all well separated in one run by the multiple heart-cutting chiral-chiral 2D-LC system.
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http://dx.doi.org/10.1016/j.chroma.2019.460845DOI Listing
May 2020

Screening of inhibitors against histone demethylation jumonji domain-containing protein 3 by capillary electrophoresis.

J Chromatogr A 2020 Feb 19;1613:460625. Epub 2019 Oct 19.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Lu, Shanghai 200032, PR China; School of physical science and technology, ShanghaiTech University, Haike Road 100, Shanghai 200120, PR China. Electronic address:

Jumonji domain-containing proteins (JMJDs) play an important role in the epigenetic regulation of gene expression. Aberrant regulation of histone modification has been observed in the progression of a variety of diseases, such as neurological disorders and cancer. Therefore, discovery of selective modulators of JMJDs is very attractive in new drug discovery. Herein, a simple capillary electrophoresis (CE) method was developed for screening of inhibitors against JMJD3. A known JMJD3 inhibitor GSK-J1, 5-carboxyfluorescein labeled substrate peptide with an amino acid sequence of KAPRKQLATKAARK(me3)SAPATGG (truncated from histone H3), as well as a small chemical library composed of 37 purified natural compounds and 30 natural extracts were used for method development and validation. The separation of substrate from its demethylated product was achieved by addition of polycation hexadimethrine bromide (HDB) in the running buffer. The enzyme activity was thus assayed accurately through separating the demethylated product from the substrate and then measuring the peak area of the product. The enzyme inhibition can be read out by comparing the peak area of the demethylated product obtained in the present of inhibitors and that of the negative control in the absence of any inhibitor. The merit of the method is proved by discovering two new JMJD3 inhibitors: salvianic acid A and puerarin 6''-O-xyloside.
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http://dx.doi.org/10.1016/j.chroma.2019.460625DOI Listing
February 2020

Targeting USP9x/SOX2 axis contributes to the anti-osteosarcoma effect of neogambogic acid.

Cancer Lett 2020 01 9;469:277-286. Epub 2019 Oct 9.

Hongqiao International Institute of Medicine, Shanghai Tongren Hospital / Faculty of Basic Medicine, Chemical Biology Division of Shanghai Universities E-Institutes, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. Electronic address:

SOX2 has been viewed as a critical oncoprotein in osteosarcoma. Emerging evidence show that inducing the degradation of transcription factors such as SOX2 is a promising strategy to make them druggable. Here, we show that neogambogic acid (NGA), an active ingredient in garcinia, significantly inhibited the proliferation of osteosarcoma cells with ubiquitin proteasome-mediated degradation of SOX2 in vitro and in vivo. We further identified USP9x as a bona fide deubiquitinase for SOX2 and NGA directly interacts with USP9x in cells. Moreover, knockdown of USP9x inhibited the proliferation and colony formation of osteosarcoma cells, which could be rescued by overexpression of SOX2. Consistent with this, knockdown of USP9x inhibited the proliferation of osteosarcoma cells in a xenograft mouse model. Collectively, we identify USP9x as the first deubiquitinating enzyme for controlling the stability of SOX2 and USP9x is a direct target for NGA. We propose that targeting the USP9x/SOX2 axis represents a novel strategy for the therapeutic of osteosarcoma and other SOX2 related cancers.
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http://dx.doi.org/10.1016/j.canlet.2019.10.015DOI Listing
January 2020

Second messenger ApA polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells.

Nat Commun 2019 10 11;10(1):4664. Epub 2019 Oct 11.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.

Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger ApA binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by ApA remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that ApA specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of ApA. These results highlight a direct polymerization signaling mechanism by the second messenger.
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http://dx.doi.org/10.1038/s41467-019-12710-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789022PMC
October 2019

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells.

Cancer Invest 2019 11;37(6):242-252. Epub 2019 Jul 11.

a Faculty of Basic Medicine, Chemical Biology Division of Shanghai Universities E-Institutes, Hongqiao International Institute of Medicine, Shanghai Tongren Hospital, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine , Shanghai , China.

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34 progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth . In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells and .
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http://dx.doi.org/10.1080/07357907.2019.1630633DOI Listing
August 2019

Determining the affinity of anti-mitotic compounds binding to colchicine binding site of tubulin by affinity probe capillary electrophoresis.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Jul 14;1121:66-71. Epub 2019 May 14.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis,Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China; School of Physical Science and Technology, ShanghaiTech University, Haike Road 100, Shanghai 200120, China. Electronic address:

The colchicine binding site of tubulin is often used to screen the anti-mitotic compounds, which are widely used as anti-cancer therapies. In the present work, an affinity probe capillary electrophoresis (APCE) method was developed for determining the affinity of anti-mitotic compounds. To this end, a fluorescently labeled affinity probe, 5-carboxyfluorescein-colchicine (F-colchicine), was prepared for the affinity competition experiment. The probe can form a stable complex with tubulin with the binding stoichiometry of 0.75, and the dissociation constant K of the complex was determined as 5.7 × 10 mol/L. In the affinity competition experiment, F-colchicine was incubated with tubulin and the test compound in the solution. The F-colchicine-tubulin complexes and free F-colchicine were quickly separated by CE and the concentration of free F-colchicine was accurately determined with the laser induced fluorescence detection. The affinity constant of the tested compound can be measured with the affinity competition binding curve. The enantiomers of the anti-mitotic compound were evaluated by using the method. The binding affinity of the enantiomers displayed an enantioselective manner. Compared to other affinity binding assay methods, our method is more straightforward, more accurate, and more cost-effective.
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http://dx.doi.org/10.1016/j.jchromb.2019.05.010DOI Listing
July 2019

Novel Triapine Derivative Induces Copper-Dependent Cell Death in Hematopoietic Cancers.

J Med Chem 2019 03 18;62(6):3107-3121. Epub 2019 Mar 18.

Shanghai Advanced Research Institute , Chinese Academy of Sciences , Shanghai 201210 , China.

Triapine, an iron chelator that inhibits ribonucleotide reductase, has been evaluated in clinical trials for cancer treatment. Triapine in combination with other chemotherapeutic agents shows promising efficacy in certain hematologic malignancies; however, it is less effective against many advanced solid tumors, probably due to the unsatisfactory potency and pharmacokinetic properties. In this report, we developed a triapine derivative IC25 (10) with potent antitumor activity. 10 Preferentially inhibited the proliferation of hematopoietic cancers by inducing mitochondria reactive oxygen species production and mitochondrial dysfunction. Unlike triapine, 10 executed cytotoxic action in a copper-dependent manner. 10-Induced up-expression of thioredoxin-interacting protein resulted in decreased thioredoxin activity to permit c-Jun N-terminal kinase and p38 activation and ultimately led to the execution of the cell death program. Remarkedly, 10 showed good bioavailability and inhibited tumor growth in mouse xenograft models. Taken together, our study identifies compound 10 as a copper-dependent antitumor agent, which may be applied to the treatment of hematopoietic cancers.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01996DOI Listing
March 2019

Preparation of a monolithic column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction for capillary liquid chromatography.

J Sep Sci 2019 Feb 4;42(3):662-669. Epub 2019 Jan 4.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Shanghai, P. R. China.

A monolithic capillary column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in-situ copolymerization of a homemade monomer N,N-dimethyl-N-acryloxyundecyl-N-(3-sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed-mode stationary phase. It was demonstrated that the mixed-mode stationary phase displayed typic dual retention mechanisms of reversed-phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.
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http://dx.doi.org/10.1002/jssc.201800826DOI Listing
February 2019

Screening of break point cluster region Abelson tyrosine kinase inhibitors by capillary electrophoresis.

J Chromatogr A 2018 Feb 8;1537:128-134. Epub 2018 Jan 8.

State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China; ShanghaiTech University, Haike Road 100, Shanghai 201210, China. Electronic address:

In the present study, a capillary electrophoresis (CE) method was developed for screening of inhibitors against the break point cluster region Abelson tyrosine kinase (BCR-ABL). The screening method was established by using 5-carboxyfluorescein labeled peptide substrate of BCR-ABL (F-ABLS), a known BCR-ABL tyrosine kinase inhibitor dasatinib, as well as a small chemical library consisting of 37 natural products. Thus, the inhibition of BCL-ABL kinase by small inhibitors was assayed by a CE system equipped with the laser induced fluorescence detector. The yield of phosphorylated product could be precisely measured through the separation by CE. The method is competent for enzymatic inhibition assay as well as the measurement of the inhibition kinetics. For screening BCR-ABL tyrosine kinase inhibitors, the hits were readily identified once the peak area of the phosphorylated products was reduced in comparison with the negative control. By taking the advantage of the screening method, luteolin and epicatechin gallate were discovered as the new BCR-ABL inhibitors.
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http://dx.doi.org/10.1016/j.chroma.2018.01.019DOI Listing
February 2018

Quantitative analysis of antithrombin III binding site in low molecular weight heparins by exhausetive heparinases digestion and capillary electrophoresis.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Nov 6;1068-1069:78-83. Epub 2017 Oct 6.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China; School of Physical Science and Technology, ShanghaiTech University, Haike Road 100, Shanghai 200031, China. Electronic address:

The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method.
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http://dx.doi.org/10.1016/j.jchromb.2017.08.047DOI Listing
November 2017

Determination of the stereoisomeric impurities of sitafloxacin by capillary electrophoresis with dual chiral additives.

J Chromatogr A 2017 Jul 7;1506:120-127. Epub 2017 May 7.

State key laboratory of Bioorganic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China; School of Physical Science and Technology, ShanghaiTech University,100 Haike Road, Shanghai, 201210, China. Electronic address:

Because of the bioactivity against the human topoisomerase II, the stereoisomeric impurities of sitafloxacin should be controlled in the process of manufactory. In the present work, a capillary electrophoresis (CE) method was developed and validated for simultaneous determination of three stereoisomeric impurities of sitafloxacin. The separation with high resolution not only for the separation of enantiomers, but also for the separation of diastereoisomers was achieved by using a background electrolyte composed of dual chiral selectors, namely γ-cyclodextrin (γ-CD) and Cu-d-phenylalanine (D-Phe) complex. The combination of two chiral selectors is indispensable to gain a high separation selectivity due to the cooperativity effect of different chiral discrimination modes: inclusion complexation (γ-CD) and ligand exchange (Cu-d-Phe). The concentrations of γ-CD, Cu and D-Phe were found to be critical to the separation. Because two chiral selectors were involved in the enantiomer separation system, multiple factors and their interaction should be simultaneously optimized by using the response surface methodology (RSM) with a face centred central composite design (FCCD). The obtained optimized separation conditions were as follows: 15mmol/L dipotassium hydrogenphosphate solution (pH 4.5) containing 15mmol/L D-Phe, 20mmol/L CuSO and 20mmol/L γ-CD, separation voltage 15kV. The method was then validated and the robustness of the method was tested. Under the optimized conditions, as low as 0.1% (m/m) stereoisomeric impurities of sitafloxacin can be determined by the method.
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http://dx.doi.org/10.1016/j.chroma.2017.05.010DOI Listing
July 2017

Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.

Anal Chem 2016 08 28;88(16):8050-7. Epub 2016 Jul 28.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences , Lingling Road 345, Shanghai 200032, China.

A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors.
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http://dx.doi.org/10.1021/acs.analchem.6b01430DOI Listing
August 2016

[Enantioseparation of ferroence derivatives with high performance liquid chromatography].

Se Pu 2016 Jan;34(1):57-61

The method for the enantioseparation of ferroence derivatives, four derivatives with single chirality and three derivatives with double chiralities containing centre and face chirality, on chiral stationary phases, namely Chiralpak IC (cellulose-tris (3, 5-dichlorobenzene carbamate)) and Chiralpak IE3 (amylose-tris(3,5-dichlorobenzene carbamate)), was investigated. We found that the three derivatives of the four chiral ferroence derivatives with single chirality can be baseline separated on Chiralpak IE3; another one can be baseline separated on Chiralpak IC. Meanwhile, the three chiral ferroence derivatives with double chiralities can be baseline separated on Chiralpak IC. The research shows that the both kinds of chiral stationary phases exhibited high enantiomeric recognition capability to the enantiomers of the chiral ferroence derivatives. This two chiral stationary phases exhibited complementary selectivities in the enantioseparation of chiral ferroence derivatives. This study provides a reference method for the enantioseparation of chiral ferroence derivatives.
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http://dx.doi.org/10.3724/sp.j.1123.2015.10002DOI Listing
January 2016

Preparation of phosphorylcholine-based hydrophilic monolithic column and application for analysis of drug-related impurities with capillary electrochromatography.

Electrophoresis 2016 07 25;37(12):1725-32. Epub 2016 May 25.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China.

A hydrophilic monolithic CEC column was prepared by thermal copolymerization of zwitterionic monomer 2-methacryloyloxyethyl phosphorylcholine (MPC), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2-methylpropene-1-sulfonate (MPS) in a polar binary porogen consisting of methanol and THF. A typical hydrophilic interaction LC retention mechanism was observed for low-molecular weight polar compounds including amides, nucleotides, and nucleosides in the separation mode of hydrophilic interaction CEC, when high content of ACN (>60%) was used as the mobile phase. The effect of the electrostatic interaction between the analytes and the stationary phase was found to be negligible. The poly(MPC-co-PETA-co-META or MPS) monolithic columns have an average column efficiency of 40 000 plates/m and displayed with a satisfactory repeatability in terms of migration time and peak areas. Finally, the column was successfully applied to determine the impurities of a positively charged drug pramipexole which are often separated by ion pair RP chromatography due to their high hydrophilicity. All four components can be baseline separated within 5 min with BGE consisting of ACN/20 mM ammonium formate buffer (pH 3.0; 80/20).
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http://dx.doi.org/10.1002/elps.201600066DOI Listing
July 2016

Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX.

Sci Rep 2015 Oct 26;5:15552. Epub 2015 Oct 26.

Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, China.

Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed.
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http://dx.doi.org/10.1038/srep15552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620443PMC
October 2015

Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography-tandem mass spectrometry.

J Chromatogr A 2015 Jun 5;1400:117-23. Epub 2015 May 5.

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, State Key Laboratory of Bio-organic and Natural Products Chemistry, Lingling Road 345, Shanghai 200032, China; ShanghaiTech University, Yueyang Road 319, Shanghai 200031, China. Electronic address:

A method for screening of inhibitors to epidermal growth factor receptor (EGFR) in natural product extracts with capillary electrophoresis (CE) in conjunction with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is reported. The method was established by employing 5-carboxyfluorescein labeled substrate peptide, two commercially available EGFR inhibitors OSI-744 and ZD1839, and a small chemical library consisted of 39 natural product extracts derived from the Traditional Chinese Medicines. Biochemical assay of crude natural product extracts was carried out by using CE equipped with a laser induced fluorescence detector. The CE separation allowed an accurately quantitative measurement of the phosphorylated product, hence the measurement of the enzymatic activity as well as the inhibition kinetics. The hits are identified if the peak area of the phosphorylated product is reduced in comparison with the negative control. The active constituents in the natural product extract were then identified by an assay-guided isolation with HPLC-MS/MS system. With the method, the flavonoids component of the Lycopus lucidus extract, namely quercetin and rutin were identified to be the active ingredients. Their IC50 values were determined as 0.88 μM and 10.1 μM, respectively. This result demonstrated a significant merit of our method in the identification of the bioactive compounds in natural products.
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http://dx.doi.org/10.1016/j.chroma.2015.04.055DOI Listing
June 2015

Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

J Chromatogr A 2015 Apr 16;1388:267-73. Epub 2015 Feb 16.

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, State Key Laboratory of Bio-organic and Natural Products Chemistry, Lingling Road 345, Shanghai 200032, China; ShanghaiTech University, Yueyang Road 319, Shanghai 200031, China. Electronic address:

In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.
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http://dx.doi.org/10.1016/j.chroma.2015.02.033DOI Listing
April 2015

[Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry].

Authors:
Feng Li Jingwu Kang

Se Pu 2014 Apr;32(4):369-75

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.
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http://dx.doi.org/10.3724/sp.j.1123.2013.12018DOI Listing
April 2014

Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

J Chromatogr A 2014 Sep 15;1359:84-90. Epub 2014 Jul 15.

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, State Key Laboratory of Organic and Natural Products Chemistry, Lingling Road 345, Shanghai 200032, China; ShanghaiTech University, Yueyang Road 319, Shanghai 200031, China. Electronic address:

A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.
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http://dx.doi.org/10.1016/j.chroma.2014.07.020DOI Listing
September 2014

Screening of protein kinase inhibitors in natural extracts by capillary electrophoresis combined with liquid chromatography-tandem mass spectrometry.

J Chromatogr A 2014 Apr 22;1337:188-93. Epub 2014 Feb 22.

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China; ShanghaiTech University, Yueyang Road 319, Shanghai 200031, China. Electronic address:

We report a capillary electrophoresis method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening of protein kinase inhibitors (PKIs) in natural extracts. Protein kinase A (PKA), substrate 5-carboxyfluorescein-labeled kemptide (CLK) and inhibitor H-89 were employed for the method development and validation. Enzymatic inhibition assay was performed with electrophoretically mediated microanalysis technique. Once the bioactivity of a natural extract was confirmed, an assay-guided isolation and structure elucidation using LC-MS/MS were accomplished to identify the compounds which are responsible for the observed bioactivity. Totally 33 natural extracts were screened with the method, and baicalin in the extract of Radix Scutellariae was identified to be a new PKI of PKA. This result demonstrated the practical applicability of our method in screening of PKIs from natural products.
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http://dx.doi.org/10.1016/j.chroma.2014.02.039DOI Listing
April 2014

[An assay for anti-factor Xa activity of low molecular weight heparins by high performance liquid size exclusion chromatography].

Se Pu 2013 Jul;31(7):684-90

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.

The "gold standard" assay for monitoring low molecular weight heparins (LMWHs) activity is the chromogenic-based anti-factor Xa assay. The methodology of an anti-factor Xa assay is that LMWH is added to a known amount of excess factor Xa and excess antithrombin. It will bind to antithrombin and form a triplet complex with factor Xa, inhibiting the activity of factor Xa. However, the residual factor Xa can still hydrolyze chromogenic peptide substrate, releasing the chromophore for photometric detection. The absorbance is inversely proportional to the amount of heparin/LMWH. The results are given in anticoagulant concentration in units/ mL of anti-factor Xa, such that high values indicate high levels of anticoagulation and low values indicate low levels of anticoagulation. Herein, a novel assay method for anti-FXa activity of LMWHs using high performance liquid size exclusion chromatography (SEC) is reported, in which antithrombin III (AT Ill ) was diluted by the buffer solution contained LMWHs. Subsequently, exogenous FXa and p-nitroaniline coupled peptide substrate were added and incubated for a period, separately. The resulting mixture was separated based on size by SEC, and the free chromophore p-nitroaniline can be detected at an absorption maximum of 385 nm without interference from the absorbance of p-nitroanilide substrates. Moreover, the measurements are not influenced by sample opacity or turbidity, so it is possible to test various complex samples, such as plasma. The assay is robust, sensitive, and cost effective.
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http://dx.doi.org/10.3724/sp.j.1123.2013.04029DOI Listing
July 2013

Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection.

Se Pu 2013 Jul;31(7):646-55

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.

A method that can be used for screening protein kinase inhibitors (PKIs) and simultaneously assessing their selectivity is described. The method is based on simultaneously assaying multiple cellular protein kinases by performing capillary electrophoresis (CE) separation and measuring the peak areas of the phosphorylated substrate peptides. The powerful separation capability of CE combined with the highly sensitive and selective laser-induced fluorescence (LIF) detector enables the direct screening of PKIs against cell lysates, which are used as an inexpensive source of enzymes. Four cell lines, three specific substrate peptides labeled with 5-carboxyfluorescein (5-FAM), two relative specific PKIs (TBB and H-89) and one non-specific PKI (staurosporine) were utilized to prove the methodology. With this method, the inhibitory activity of the tested compounds against multiple protein kinases was identified in parallel by comparing the peak areas of the phosphorylated substrates with those obtained in the absence of any inhibitors. The reduced peak area of the phosphorylated substrate definitively represents a positive screening result. Simultaneously, assaying the inhibition of one inhibitor against mutiple cellular protein kinases enables the assessment of its selectivity. Compared to the conventional, single-target screening format, the cell lysate-based multi-target method is more informative, more straightforward and more cost effective.
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http://dx.doi.org/10.3724/sp.j.1123.2013.04032DOI Listing
July 2013

[Screening of the active ingredients in natural products by capillary electrophoresis and high performance liquid chromatography-mass spectrometry].

Se Pu 2013 Jul;31(7):640-5

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.

A new strategy for screening the crude natural extracts and quickly identifying the bioactive compounds was developed. In combination with high performance liquid chromatography-mass spectrometry (HPLC-MS) , the biologically active compounds, such as the enzyme i n the crude natural extract ca n be quickly identified by capillary electrophoresis (CE) -based activity assay. The crude natural extracts were assayed by a CE-based enzyme inhibitor screening method, and the active extract was isolated by HPLC-MS/MS with a semipreparative column. Then, each eluted component was assayed again with the CE-based assay method. Finally, the structures of the identified active compounds were elucidated by MS/MS analysis. Acetylcholinesterase ( ACHE), its substrate acetylthiocholine chloride ( AThCh), as well as the crude extract of Rhizoma coptidis were utilized for the proof of the methodology. Seven isoquinoline alkaloids, namely jatrorrhizine, epiberberine, columbamine, coptisine, corysamine, palmatine and berberine were identified to be active as the inhibitors of ACHE. Their IC50 values were 40, 442, 38, 182, 419, 54 and 16 micromol/L, respectively. Compared with the traditional screening methods, the method is characterized with several advantages, such as extremely low sample and reagent consumption, high speed of analysis, high sensitivity of detection, high throughput in terms of preparation of the natural products by HPLC. Overall, the results demonstrate that the method is valuable for the screening of the bioactive compounds in the crude natural extracts.
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http://dx.doi.org/10.3724/sp.j.1123.2013.03013DOI Listing
July 2013

Enzyme inhibitor screening by CE with an on-column immobilized enzyme microreactor created by an ionic binding technique.

Methods Mol Biol 2013 ;984:321-7

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.

Enzymes are an important class of drug targets. In the early stage of the drug discovery, the major task is to find out the inhibitors of a given enzyme target in a compound library. Herein we describe a method for screening the enzyme inhibitors in the complex mixtures (such as the natural extracts) by capillary electrophoresis with an on-column immobilized enzyme microreactor. The enzyme molecules are immobilized on the capillary wall via ionic binding with the positively charged coating, which was created by simply flushing the column with a solution of polyelectrolyte hexadimethrine bromide. The activities of the immobilized enzymes are assayed by performing the electrophoretic separation and thereafter determining the product of the enzyme-mediated reaction. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison with that in the reference electropherogram obtained in the absence of any inhibitor.
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http://dx.doi.org/10.1007/978-1-62703-296-4_23DOI Listing
July 2013

Use of macrocyclic antibiotics as the chiral selectors in capillary electrophoresis.

Methods Mol Biol 2013 ;970:307-17

Chinese Academy of Sciences, Shanghai Institute of Organic Chemistry, Shanghai, China.

Macrocyclic antibiotics, especially vancomycin, represent a class of versatile chiral selectors for enantioseparations by capillary electrophoresis (CE). In this chapter, we describe the protocol for performing CE enantioseparations with vancomycin as the chiral selector. Dynamic coating of the capillary with polymers including the positively charged polyelectrolyte hexadimethrine bromide or electro-neutral poly(dimethylacrylamide) proved to be very useful in order to reduce the adsorption of vancomycin onto the capillary wall resulting in an improved separation efficiency. The partial filling technique is employed for improvement of the detection sensitivity. Utilization of these techniques makes the CE enantioseparation with vancomycin more practical and robust.
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http://dx.doi.org/10.1007/978-1-62703-263-6_20DOI Listing
June 2013

Structural analysis of low molecular weight heparin by ultraperformance size exclusion chromatography/time of flight mass spectrometry and capillary zone electrophoresis.

Anal Chem 2013 Feb 17;85(3):1819-27. Epub 2013 Jan 17.

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Lingling Road 345, Shanghai 200032, China.

Although low molecular weight heparins (LMWHs) have been used as anticoagulant agents for over 2 decades, their structures have not been fully characterized. In this work, we propose a new strategy for the comprehensive structural analysis of LMWHs based on the combination of ultraperformance size exclusion chromatography/electrospray quadruple time-of-flight-mass spectrometry (UPSEC/Q-TOF-MS) and capillary zone electrophoresis (CZE). More than 70 components, including oligosaccharides with special structures such as 1,6-anhydro rings, saturated uronic acid at the nonreducing end and odd-numbered saccharides units were identified with UPSEC/Q-TOF-MS. Furthermore, a more detailed compositional analysis was accomplished by CZE analysis. PEG10000 and MgCl(2) were added to the background electrolyte to separate those saccharides with the nearly same charge-to-mass ratio. Baseline separation and quantification of all the building blocks of the most complex LMWH, namely, enoxaparin, which include 10 disaccharides, 1 trisaccharide, 2 tetrasaccharides, and, of particular importance, 4 1,6-anhyro derivatives, was achieved using CZE for the first time. Additionally, the peaks of oligosaccharides, in the absence of commercially available standards, were assigned on the basis of the linear correlation between the electrophoretic mobilities of oligosaccharides and their charge-to-mass ratios. These two approaches are simple and robust for structural analysis of LMWHs.
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http://dx.doi.org/10.1021/ac303185wDOI Listing
February 2013

Self-resistance to an antitumor antibiotic: a DNA glycosylase triggers the base-excision repair system in yatakemycin biosynthesis.

Angew Chem Int Ed Engl 2012 Oct 17;51(42):10532-6. Epub 2012 Sep 17.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Chinese Academy of Sciences, China.

Resistance is (not) futile: The yatakemycin biosynthetic gene cluster involves the ytkR2 gene, which encodes a protein with homology to a recently discovered bacterial DNA glycosylase. Genetic validation in vivo, biochemical assays, and in vitro mutagenesis studies revealed that YtkR2 confers resistance for the bacteria by specifically recognizing and cleaving the YTM-modified base (see scheme).
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http://dx.doi.org/10.1002/anie.201204109DOI Listing
October 2012