Research On Synthesis And Antitumor Activity Of Proteolysis Targeting Chimeras Targeting Mutant EGFR Based On Lapatinib

Epidermal growth factor receptor is a popular target in the field of targeted food development and cancer chemoprophylaxis.In order to inhibit the overexpression of epidermal growth factor receptor（EGFR）,natural functional food factors,small molecule EGFR inhibitors and monoclonal antibodies have received extensive attention as effective strategies.However,its application is limited by its high effective concentration,poor bioavailability and off-target mutations.In recent years,the emergence of Targeted protein degradation（TPD）has provided a new option for cancer treatment.The core of this technology is to induce target protein reduction or consumption by hijacking the ubiquitin-protease system.Among them,Proteolysis Targeting Chimeras（PROTACs）,a relatively mature technology,aims to effectively target protein degradation by synthesizing a bifunctional molecule which on the one hand targets the target protein and on the other hand hijacks the ubiquitination degradation system.In this study,the self-designed Lapatinib derivative was used as the target protein-binding ligand,and 6 different long-chain molecules were used to bind 3 E3ligand.Finally,18 PROTACs molecules were obtained.PROTACs effectively degraded mutant EGFR proteins(EGFR^L858R/T790M and EGFR^e19d)in NCI-H1975 and HCC-827 cells.The DC₅₀ values of compounds 32 and 33 with the best degradation effect on EGFR^L858R/T790M protein were 63.02 n M and 986.5 n M,respectively.Both compounds successfully degraded EGFR protein and inhibited the expression of its downstream signaling pathway protein,and the inhibitory effect was far greater than Lapatinib.The results of follow-up experiments showed that compound 32 and 33showed effective anti-proliferative activity against HCC-827 cell lines,with IC₅₀values of 695.4 n M and 972.2 n M,respectively.Compounds 32 and 33 showed effective antiproliferative activity against NCI-H1975 cell lines,with IC₅₀ values of358.7 n M and 735.9 n M,respectively.The results of competitive experiments showed that after Osimertinib and lenalidomide,as competitive inhibitors,are used to preincubate with NCI-H1975 cells respectively,the degradation effect of EGFR^L858R/T790M protein was reduced by produced by NCI-H1975 cells treated with compounds 32 and 33.This effect may be caused by the competition between Osimertinib and lenalidomide with PROTACs for the protein active sites of CRBN E3ligase and EGFR^L858R/T790M,respectively.In addition,after the addition of 26S protease inhibitor（MG-132）and NCI-H1975 cells for pre-incubation,compounds 32and 33 on EGFR^L858R/T790M protein degradation also showed a decreasing trend.This indicated that the EGFR^L858R/T790M protein degradation of NCI-H1975 cells after PROTACs treatment was related to 26S protease.Further the results of molecular simulation experiments showed that the PROTACs molecule,which was composed of the appropriate linking groups,target protein ligand and E3 ligand,could promote the formation of CRBN E3 ligase-PROTACs-EGFR^L858R/T790M ternary complex.This stable ternary complex is the molecular basis of targeted protein degradation.In conclusion,this study successfully obtained PROTACs that can target the degradation of mutant EGFR protein,which showed high antitumor activity in vitro and improved the defect of poor inhibition effect of Lapatinib on mutant EGFR.The results of this study not only provide some design ideas and experimental methods for the development of PROTACs targeting mutant EGFR,but also provide research ideas for the molecular design of PROTACs based on food functional factors.