Screening And Identifying The Inhibitors Of Src Protein Tyrosine Kinase From Hedyotis Diffusa Willd

Tyrosine kinase is a sort of protein that can phosphorylate the tyrosine amino acid of many key proteins in intracellular signal transduction pathways, which involved in cell migration, growth, differentiation and survival. Abundance researches indicate abnormal activation of tyrosine kinase is closely implicated in the development of many tumors. At present, inhibition of tyrosine kinases is a focus of research on anti-cancer medicine. Till now, over ten kinds of tyrosine kinase inhibitors and antibodies have been in I～III phases of clinical test, and some of them have come into the market, e.g. Gleevec (STI571) against the target of protein Bcr-Abl/PDGFR, Iressa (ZD1839) and Erlotinib (OSI-774) against the target of protein EGFR.In this paper, it is studied in the screening of Src tyrosine kinase inhibitors in Hedyotidis Diffusae willd, a Chinese herb in common use in cancer therapy, and two anthraquinones with Src tyrosine kinase inhibitory ability were picked out. The inclusion-body protein of GST-v-Src was bacterially expressed and collected, using the engineering bacterial E.coli. BL21 (DE3) pGEX-KT/v-src containing v-src gene fraction, and then, was actived by denature and renature. A model system for the rapid screening of Src tyrosine kinase inhibitors was established in vitro, in which the activity of protein GST-v-Src and inhibitory ability toward Src tyrosine kinase was assayed by ELISA: Use a poly Glu:Tyr (4:1) as the substrate, and the fraction of phosphorylated substrate is visualized using a phosphotyrosine monoclonal antibody conjugated to HRP (HRP-PY20). Two component having Src tyrosine kinase inhibitory ability was isolated and purified from Hedyotidis Diffusae willd by Silica gel column chromatography and silica TLC fractionation. The structures of the two anthraquinones were deduced to be 2-methyl-3-hydroxyanthraquinone and2-methoxy-3-hydroxyanthraquinone using IR, UV, MS, ¹H-NMR, ¹³C-NMR and HMQC analysis. Both of two compounds showed inhibition activity against the growth of HepG2, SPC-A-1, Bcap37 cancer cell lines.