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Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI

 Yonghyo Kim  ;  Yutaka Sugihara  ;  Tae Young Kim  ;  Sung Min Cho  ;  Jin Young Kim  ;  Ju Yeon Lee  ;  Jong Shin Yoo  ;  Doona Song  ;  Gyoonhee Han  ;  Melinda Rezeli  ;  Charlotte Welinder  ;  Roger Appelqvist  ;  György Marko-Varga  ;  Ho Jeong Kwon 
 BIOMOLECULES, Vol.10(4) : 508, 2020-03 
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Animals ; CD13 Antigens / metabolism ; Curcumin / pharmacology ; Drug Evaluation, Preclinical / methods* ; Female ; Human Umbilical Vein Endothelial Cells ; Humans ; Ibogaine / analogs & derivatives* ; Ibogaine / chemistry ; Ibogaine / pharmacokinetics ; Ibogaine / pharmacology ; Mass Spectrometry ; Mice, Inbred BALB C ; Molecular Docking Simulation ; Tissue Distribution ; Vascular Endothelial Growth Factor Receptor-2 / chemistry ; Vascular Endothelial Growth Factor Receptor-2 / metabolism* ; Xenograft Model Antitumor Assays
target identification ; target validation ; label-free method for drugs ; anti-angiogenesis ; mechanism of action ; receptor tyrosine kinases ; curcumin ; natural products
Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.
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1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
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