Study On The Interaction Mechanism Between Small Molecule Inhibitors And EGFR Kinase

The epidermal growth factor receptor(EGFR)is an important research target for small molecule inhibitors of tumors.In the previous experiments,our group designed and synthesized a large number of compounds by the method of “active substructure combination”.In this study,we tried to figure out the compounds with high inhibitory activity by using high EGFR expression cell lines,further to evaluate the compond's effect on EGFR at the protein level.Finally,we investigated the effect of compounds on the activity of EGFR and downstream pathway of EGFR in lung cancer cells and found out the mechanism of action.We assessed the antiproliferation of two series of compounds by MTT method against A549,PC-3,Hep G2 and SMMC-7721 with high EGFR expression level,K562 with weak EGFR expression level,and the nornal cell line NRK-52 E.The C series derived from gefitinib showed good activity in A549 and PC-3 cells,and the WQ series derived from solafenib showed excellent activity in A549,PC-3,SMMC-7721 and K562 cells.The activity of NRK-52 E cell line was been further assessed by MTT assay and the results showed that C-19 and W-18 had little effect on the activity of NRK-52 E cell.Then we evaluated the cell proliferation effects of compounds,and performed colony formation on the A549 and PC-3 cell lines.The results of colony formation showed that the colonies presented significant corresponding down regulated trends on A549 and PC-3 cells when stimulated by a range of conpounds doses(1.25,2.5,5,10 and 20 ?M).To investigate the cell migration of compounds,we performed wound healing on the A549 and PC-3 cell lines.The results showed that C-19 and W-18 had significant effect on the migration ability on A549 and PC-3.In order to assess the effects of different concentrations of compounds on apoptosis,we performed Hoechst 33258 staining on the A549 and PC-3 cell lines.The result showed that it was easy to observe the distinct pattern of morphological changes,including cell shrinkage,fragmentation of the nucleus,and chromatin condensation after treating with different concentrations of C-19 and W-18 for 24 hours on A549 and PC-3 cells.For assessing the effects of compounds on apoptosis,the Annexin VFITC / PI double staining method was been used to detect the apoptosis of cells by flow cytometry.Thus,the results showed that with the increase of concentration,the rate of apoptosis increased,and there was significant apoptosis in low concentration cells.The inhibition effect of EGFR expression was tested by ELISA in vitro,and the data proved that the inhibition activity of W-18 on A549 and PC-3 cell lines was better than that of the control sorafenib,and C-19 showed better activity as well.To detect the effect of compounds on the expression of EGFR and downstream PI3 K / Akt,we used the western blot assay.Western blot results showed that both C-19 and W-18 could down-regulate the expression of p-EGFR(Tyr1068)and EGFR.They can also down-regulate the expression of key proteins AKT and p-AKT(Ser473)of downstream signaling pathway.In order to evaluate these promising compounds and to guide structure-activity relationship(SAR)studies,Surflex-Dock model was established by using Sybyl-x2.0 software based on the antitumor activities of compounds.The results indicated that C-19 with EGFRwt or EGFRT790 M / L858 R had well link through hydrogen bond connection.The binding score of compound W-18 and EGFRwt is low,which may be due to the less hydrogen bond effect.The binding ability with EGFRT790 M/L858 R is enhanced because of the strengthen hydrogen bond link.The research of this subject confirmed that compounds C-19 and W-18 inhibited the activities of the proliferation,migration,and invasion of several tumor cells.Specially,they are selective for EGFR overexpressing cell lines A549 and PC-3.It could affect expression of EGFR kinase and the downstream of EGFR PI3K/Akt.