Alkaline Tyrosine Kinase Inhibitors Promote The Accumulation Of P62 In Tumor Cells And The Study On The Design,Synthesis And Activity Evaluation Of P62 PROTAC

Background:Targeted tumor therapy has developed rapidly in the past 30 years,but at the same time,drug resistance in molecular targeted tumor therapy is becoming more and more prominent.Recently,it has been reported that lysosomes are widely involved in the formation of drug resistance to a variety of molecular targeted drugs(EGFR inhibitors,VEGFR inhibitors,etc.).In previous studies,we have found that basic EGFR TKIs such as Gefitinib and AZD9291 can cause functional blocking of autophagy lysosome and accumulation of oncogenic protein p62,eventually leading to the resistance of tumor cells to EGFR TKIs such as Gefitinib and AZD9291,and preliminarily confirmed that the alkaline neutralization and modification strategy is preliminarily proved to be a potential rational modification strategy for EGFR TKIs.Objective:The scope of EGFR TKIs was extended to most of the TKIs to analyze whether basic TKIs could induce the accumulation of p62.In addition,a class of alkaline-neutralizing analogs with strong acidic groups were designed and synthesized in order to further reduce the molecular alkalinity of EGFR TKIs leading to its promotion of tumor cell p62 accumulation,and further improve the anti-NSCLC activity of EGFR TKIs.Moreover,novel p62 PROTAC molecules were designed and synthesized to provide effective drug candidates for the treatment of various diseases with abnormal activation of p62.Method:Western blot was used to analyze the effect of other basic TKIs on the accumulation of p62.According to the eutectic structure,the basic neutralization analogs of TKIs were reasonably designed and synthesized.Western blot was used to analyze the reversal effect of alkaline neutralization strategy on the accumulation of p62 in tumor cells induced by basic TKIs.The anti-NSCLC proliferative activities of Gefitinib,Osimertinib and their alkaline neutralizing analogs were detected by MTS assay.The inhibitory activities of Gefitinib,Osimertinib and their alkaline neutralizing analogues on EGFR signaling pathway were analyzed by Western blot.Biotin-labeled assay verifies that p62-ZZi directly binds to p62 protein.A series of p62 candidate PROTAC molecules were designed and synthesized.The degradation of p62 by p62 candidate PROTAC molecule was analyzed by Western blot.Results:Basic TKIs can induce the accumulation of p62 in tumor cells.Through chemical modification,alkaline neutralization analogs can reverse the accumulation of p62 caused by TKIs,but the weakened kinase inhibitory activity leads to the failure of alkaline neutralization modification to sensitization of TKIs.p62-ZZi has been reported as a potential p62-binding small molecule,which can be used in the design of p62 PROTAC molecules.Several candidate P62 PROTAC small molecules were designed and synthesized,but the degradation activity of the obtained PROTAC molecules was not ideal.However,the implementation of this study has important reference significance for the development of p62 PROTAC molecule.Conclusion:Although we did completely alter the inhibition of lysosomes by EGFR TKIs and the accumulation induction of p62 by a completely alkaline neutralization and reversal strategy.However,changes in the lipid solubility of alkaline-neutralizing analogues significantly impair the ability of analogues to enter cells,resulting in a significant decrease in proliferation-inhibiting activity.In this study,it is clear that p62-ZZi can be used as the ligand part of the target protein of PROTAC,and a mature experimental system for the synthesis of p62 PROTAC candidate molecules is established,which lays a solid foundation for the subsequent synthesis of PROTAC.