Screening And Structure–Activity Relationship (SAR) Study Of The Protac-Based Degraders For Target Therapy In Cancers

Background: Proteolysis Targeting Chimera(PROTAC)is a bifunctional chimeric molecule that can bind both the target protein and E3 ligase,so that the target protein can be labeled by ubiquitination,and enters in the proteasome pathway for degradation.A PROTAC has three components: ligand for target protein,ligand for E3 ubiquitin ligase,and a linker between the two.In this project,we synthesized and screened a series of PROTAC molecules targeting CDK4/6 or EGFR,respectively.The goal of the project is to identify the PROTAC that can degrade the target proteins efficiently proteins,therefore provide the candidate compound leads for cancer tumor targeted therapeutics.Methods: The PROTACs ZM7,ZM8,ZM9,synthesized based on palbociclib,a CDK4/6 inhibitor for breast cancer target therapy,were used to treat the breast cancer cells or acute lymphocytic leukemia cells HAL-01 at different doses for 72 h.CCK8 reagent was used to measure the cell viability;and Western blot was used to detect the protein levels of CDK4 and CDK6,as well as cyclin D,Rb and phospho-Rb proteins in the pathway of CDK4/6.The PROTACs ZM3,ZM4,ZM5,ZM6,based on Gefitinib,the marketed drug EGFR tyrosine kinase inhibitor(TKI),were used to treat NSCLC cells at various concentrations for 72 h.Western blot was used to detect the degradation of EGFR.The PROTACs ZME6,ZME8,ZM11,ZME12 based on the EGFR allosteric inhibitor EAI045 derivative,were used to treat NSCLC cells at different dilutions for 72 h.CCK8 reagent was used to detect the cell viability and Western blot was used to detect the degradation of EGFR.Results: ZM7,ZM8 and ZM9 inhibited the proliferation of 5 different breast cancer cell lines T-47 D,MCF-7,MDA-MB-468,MDA-MB-231,ZR-75-1 and acute lymphoblastic leukemia cells HAL-01 in a concentration dependent manner.However,ZM7,ZM8 and ZM9 did not degrade CDK4 and CDK6 in breast cancer cell lines;while ZM8 and ZM9 partially degraded CDK6 in HAL-01.ZM3,ZM4,ZM5 and ZM6 inhibited the proliferation of Gefitinib-resistant and Gefitinib-sensitive NSCLC cells,and the inhibitory effects on the sensitive cells was more significant than the resistant cells.ZM4,ZM5 and ZM6 showed a dose-dependent degradation of EGFR in the sensitive cells PC-9 and HCC827,but not in the resistant cells PC-9-IR,H1975 and EGFR wild-type cell A549.ZME6,ZME8 and ZME11 had no inhibitory effect on the proliferation of NSCLC cells,but ZME12 inhibited cell proliferation at higher concentration.ZME6,ZME8 and ZME11 can significantly degrade EGFR at 5h.Conclusion: ZM7,ZM8 and ZM9 did not degrade CDK4 and CDK6 in breast cancer cell lines,while ZM8 and ZM9 could partially degrade CDK6 in HAL-01.ZM4,ZM5 and ZM6 showed a dose-dependent degradation of EGFR in the Gefitinib sensitive cells PC-9 and HCC827,but not in resistant cells PC-9-IR,H1975 and EGFR wild-type cell A549.The PROTACs with EAI045 derivative as warhead,ZME6,ZME8 and ZME11,can degrade EGFR significantly at 5h.