KIT-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-GIST
Jingting Feng1, Yuhan Jiang1, Sihan Liu1
1School of Pharmacy, Xi'an Jiaotong University, 76# Yanta West Road, Xi'an, 710061, China.
Abstract:
Gastrointestinal mesenchymal tumors, as the most common mesenchymal tumors in the gastrointestinal tract, are adjuvantly treated with multi-targeted tyrosine kinase inhibitors, such as imatinib and sunitinib, but there are problems of drug resistance and complex methods of monitoring therapeutic agents. The pathogenesis of this disease is related to mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α, an important target for drug therapy. In recent years, the screening of relevant tyrosine kinase inhibitors from traditional Chinese medicine has become a hotspot in antitumor drug research. In the current study, the KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column was prepared with satisfying specificity, selectivity, and reproducibility by chemically bonding high KIT expression cell membranes to the silica gel surface using the SNAP-tag technology. The KIT-SNAP-tag/CMC-HPLC-MS two-dimensional coupling system was investigated using the positive drug imatinib, and the results showed that the system was a reliable model for screening potential antitumor compounds from complex systems. This system screened and identified three potential active compounds of evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO), which possibly target the KIT receptor, from the alcoholic extract of the traditional Chinese medicine Evodia rutaecarpa. Then, the KD values of the interaction of EVO, RUT, and DEVO with KIT receptors measured using nonlinear chromatography were 7.75 (±4.93) × 10-6, 1.42 (±0.71) × 10-6, and 2.34 (±1.86) × 10-6 mol/L, respectively. In addition, the methyl thiazolyl tetrazolium assay validated the active effects of EVO and RUT in inhibiting the proliferation of high KIT-expressing cells in the ranges of 0.1-10 µmol/L and 0.1-50 µmol/L, respectively. In conclusion, the KIT-SNAP-tag/CMC could be a reliable model for screening antitumor components from complex systems.
Insights
Researchers developed a KIT-SNAP-tag/CMC system to screen traditional Chinese medicines for gastrointestinal mesenchymal tumor treatments. This method identified three compounds, evodiamine, rutaecarpin, and dehydroevodiamine, that target the KIT receptor and inhibit cancer cell growth.
Area of Science:
- Biochemistry
- Pharmacology
- Traditional Chinese Medicine
Background:
- Gastrointestinal mesenchymal tumors (GIMTs) are common and treated with tyrosine kinase inhibitors (TKIs) like imatinib.
- Drug resistance and complex monitoring limit current TKI therapies for GIMTs.
- Mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α drive GIMT pathogenesis and are key drug targets.
Purpose of the Study:
- To develop a novel method for screening traditional Chinese medicine (TCM) compounds targeting the KIT receptor for GIMT treatment.
- To identify potential KIT-targeting antitumor agents from the TCM Evodia rutaecarpa.
Main Methods:
- Preparation of a KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column using SNAP-tag technology.
- Coupling KIT-SNAP-tag/CMC with high-performance liquid chromatography-mass spectrometry (HPLC-MS) for a two-dimensional screening system.
- Validation of compound activity using nonlinear chromatography for binding affinity (KD) and methyl thiazolyl tetrazolium (MTT) assays for cell proliferation inhibition.
Main Results:
- The KIT-SNAP-tag/CMC-HPLC-MS system demonstrated high specificity, selectivity, and reproducibility for screening.
- Three potential KIT-targeting compounds—evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO)—were identified from Evodia rutaecarpa extract.
- EVO, RUT, and DEVO showed varying binding affinities to KIT receptors, and EVO and RUT inhibited proliferation of high KIT-expressing cells.
Conclusions:
- The KIT-SNAP-tag/CMC system is a reliable platform for screening antitumor compounds from complex mixtures like TCM.
- Evodiamine and rutaecarpin are promising candidates for further development as targeted therapies for KIT-driven GIMTs.
- This approach facilitates the discovery of novel anticancer agents from natural products.
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