Study On The Antitumor Effect Of EGFR Inhibitors WS-157 And YS-67

The human epidermal growth factor receptor(EGFR),a receptor tyrosine kinase,is associated with the development of many types of human cancers,It is currently known that EGFR often undergoes genetic changes such as gene amplification,activation of kinase domain mutations,in-frame deletions in the extracellular domain of EGFR(EGFR vIII)to promote its abnormal activation.The first generation of EGFR inhibitors are ATP competitive reversible inhibitors,The working mechanism of first-generation EGFR TKIs is to block the activation of downstream signaling induced by EGFR through binding to the ATP-binding sites.Patients harboring oncogenic mutations in the epidermal growth factor receptor(EGFR)which constitute 10–30% of the non-small cell lung cancer(NSCLC)population resulting in an estimated 100–200 thousand newly diagnosed cases per year globally.The two frequent and mutually-exclusive primary mutations are EGFR L858 R and EGFR Del(exon 19 deletions between amino acids 746 and 750)which account for approximately 85% of all mutant EGFR NSCLC cases,Patients with these somatic mutants can be treated with first-generation EGFR kinase inhibitor drugs(gefitinib and erlotinib)which have high response rates;However,almost all patients who received gefitinib/erlotinib treatment eventually and inevitably acquired resistance,with a median progression-free survival(mPFS)of approximately 9 to 11 months,The most common mechanism of acquired resistance is point mutation of exon 20 to T790 M,with an incidence of approximately 50% to 60%.In this study,through the structural modification of the first generation of EGFR inhibitors,after the preliminary screening of the compounds,we found two compounds with better activity,WS-157 and YS-67,which were evaluated at the molecular,cellular and animal levels.Part I: Antitumor effect of EGFR inhibitor WS-157Ws-157 is a new EGFR inhibitor,which is a new quinazoline derivative based on the structural modification of gefitinib.WS-157 showed excellent inhibitory activities against EGFR wt(IC50 = 0.81nM),EGFR [d746-750](IC50 = 1.2 nM)and EGFR L858R(IC50 = 1.1 nM),which was superior to Gefitinib.Western Blot analysis in EGFR WT cells showed that WS-157 significantly inhibited the levels of EGFinduced p-EGFR and p-AKT in a dose-dependent manner,and WS-157 or Gefitinib at 1?M almost completely blocked the levels of p-EGFR and p-AKT;However,detection of the EGFR T790 M / L858 R mutant cell line H1975 revealed that the compounds WS-157 and Gefitinib did not block the levels of p-EGFR and p-AKT in this cell,further confirming the targeting of WS-157,which was only strongly inhibited by EGFR wt or sensitive mutant EGFR [d746-750] and EGFR L858R;After removal of WS-157,p-EGFR and p-AKT levels gradually recovered,This indicates that WS-157 is not only a selective inhibitor of EGFR but also a reversible inhibitor.Next,SRB assay was used to detect the proliferation inhibition of WS-157 against tumor cells of different tissue origins.WS-157 showed excellent antiproliferative activity against all tumor cells,and the IC 50 values of this compound were similar to that of gefitinib.Cell cycle detection by flow cytometry showed that WS-157 caused strong G1 phase arrest in A549 cell lines with high EGFR WT expression.WS-157 also exhibited the ability to reduce colony formation and wound healing as same as gefitinib.The anti-tumor efficacy of WS-157 in A431 xenograft tumor model in nude mice was further tested.WS-157(10 mg/kg/d)showed good anti-tumor activity,and the tumor load of nude mice was almost completely inhibited by WS-157(40mg/kg/d).These results suggest that WS-157,is a reversible and selective inhibitor of EGFR,which not only significantly inhibits the activation of EGFR and its downstream signaling pathways at the cellular level,but also significantly inhibits tumor growth at the animal level.Part II: Antitumor effect of EGFR inhibitor YS-67We obtained a new synthetic quinazoline derivative YS-67 by preliminary compounds screening,YS-67 showed excellent inhibitory activity against EGFR wt,EGFR [d746-750] and EGFR L858R(IC50= 1-10nM),which was equivalent to the inhibitory activity of Gefitinib,indicating that YS-67 is a highly selective EGFR inhibitor.Western Blot analysis in A431 cells and A549 cells with high expression of EGFR WT showed that YS-67 significantly inhibited EGF-induced phosphorylation of EGFR at 1?M concentration,with a slightly weaker inhibitory effect than gefitinib;In EGFR T790M/L858 R mutant cell line H1975,it was found that this compound,similar to gefitinib,could not block p-EGFR and p-AKT.This indicates that the compound is an inhibitor of EGFR wt or sensitive mutant EGFR [d746-750],EGFR L858 R.After the removal of YS-67 compound,its inhibition of p-EGFR and p-AKT gradually recovered,Therefore,YS-67 is not only a selective inhibitor of EGFR but also a reversible inhibitor.Further SRB assay showed that YS-67 had excellent antiproliferation activity against tumor cells from different tissue sources,and was significantly stronger than gefitinib.It was also found that YS-67 significantly inhibited the ability of cell clone formation and scratch repair.In the A431 xenograft tumor model in nude mice,we detected that YS-67(40mg/kg/d)group had an obvious inhibitory effect on tumor growth,but the inhibitory activity of YS-67 on tumor was slightly weaker than that of the positive control compound gefitinib,This further suggests that YS-67 May have other targets,which need to be further studied.The above results indicate that YS-67 identified in this study is a selective and reversible inhibitor of EGFR,but its target and anti-tumor mechanism need further study.In conclusion,our study found that compounds WS-157 and YS-67 had strong inhibitory activity against EGFR.By inhibiting the activity of EGFR kinase,WS-157 blocks its downstream signal transduction pathway,thereby inhibiting the proliferation of tumor cells,resulting in anti-tumor effects.YS-67 is also an EGFR kinase inhibitor,capable of inhibiting the phosphorylation level of EGFR and downstream molecule AKT in vitro,and has anti-tumor activity in vivo.However,YS-67 showed lower activity against EGFR kinase than Gefitinib,but stronger antiproliferation activity against tumor cells from different tissue sources than Gefitinib,indicating that there are other targets for YS-67,which needs further study.