Design,Synthesis,and Biological Evaluation Of Proteolysis-Targeting Chimeras As Degraders Of Glutathione Peroxidase 4

Objective Ferroptosis is a novel type of cell death identified in 2012 and is associated with a variety of physiological and pathological processes.GPX4,a key selenocysteinecontaining antioxidant enzyme in the GPX family,plays a key role in protecting cells from ferroptosis by inhibiting the formation of lipid peroxides.The discovery and rapid development of targeted protein degradation approaches has received increasing attention,particularly protein hydrolysis targeting chimeras(PROTACs).The aim of this study was to synthesize PROTACs to induce ferroptosis by degrading GPX4.Methods By analyzing the cocrystal structure of ML 162 in complex with GPX4(PDB ID:6HKQ)and modifying the ligand ML 162,we synthesized different types of PROTACs incorporating different linkers and ligands of the E3 ubiquitin ligase.The obtained GPX4-PROTACs were evaluated by Western blotting and the cytotoxicity was determined by the CCK-8 assay.Changes in LPO levels were measured by flow cytometric analysis.Results In this work,we designed,synthesized and evaluated different types of GPX4PROTACs using ML 162 derivatives and ligands for CRBN/VHL E3 ligases.Western blotting assays showed that most PROTACs were less efficient in degrading GPX4,and GDC-22 showed the highest degradation efficiency for GPX4(45%at 10 μM).However,no significant cytotoxicity and enhanced intracellular lipid peroxidation were observed for GDC-22 in the follow-up experiments.Meanwhile,GDC-11 showed a relatively balanced biology in terms of GPX4 degradation(33%degradation at 10 μM),cytotoxicity(IC50=11.69 μM)and lipid peroxide accumulation(2-fold increase compared to DMSO),showing a typical ferroptosis profile.Another PROTAC,GDC1,exhibited similar inhibition of HT1080 cell growth and enhanced intracellular lipid peroxidation,but did not show significant GPX4 degradation,suggesting the existence of other mechanisms of cell ferroptotic death.Given their moderate degradation of GPX4,we hypothesized that modifications to the GPX4 ligand ML 162 may not be suitable for binding to GPX4.Our hypothesis was confirmed by Molecular docking and quantum chemical theoretical calculation results.This work lays a foundation for the subsequent research of GPX4-PROTACs,and provides ideas for our group to further design and synthesize GPX4 degraders.Conclusion PROTAC is a strategy to induce ferroptosis in cells by degrading GPX4.In this study,we synthesized different types of PROTACs incorporating different linkers and ligands of the E3 ubiquitin ligase.This work lays the foundation for subsequent studies of GPX4-PROTACs and provides ideas for further design and synthesis of targeted GPX4 degraders being carried out in our group.