Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.
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.
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.
Yanmei
Y
Zhang
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.
Feng
F
Li
ShanghaiTech University, Yueyang Road 319, Shanghai 200031, China.
Mingxia
M
Li
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: jingwu.kang@sioc.ac.cn.
Jingwu
J
Kang
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000475:Alkenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001688:Biological Products / Q000494:pharmacology*; D018797:Cell Cycle Proteins; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004365:Drugs, Chinese Herbal; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D005452:Fluoresceins / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D009025:Morpholines / Q000737:chemistry / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry; D059808:Polyphenols / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D027561:Salvia miltiorrhiza / Q000737:chemistry; D054852:Small Molecule Libraries / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D001572:Benzofurans
D048868:Adaptor Proteins, Signal Transducing; D000475:Alkenes; D001688:Biological Products; D018797:Cell Cycle Proteins; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D009025:Morpholines; D010750:Phosphoproteins; D010936:Plant Extracts; D059808:Polyphenols; D054852:Small Molecule Libraries; D058570:TOR Serine-Threonine Kinases; C087000:EIF4EBP1 protein, human; C568740:salvianolic acid; C045132:4-carboxyfluorescein; C546624:(5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol; C546842:MTOR protein, human; C000597819:salvianolic acid C; D001572:Benzofurans
CE; HPLC–MS/MS; Natural products; mTOR inhibitor