CETZOLE Analogs\nas Potent Ferroptosis Inducers and\nTheir Target Identification Using Covalent/Affinity Probes

Ferroptosis\nis a recently discovered cell death mechanism triggered\nby iron-dependent elevation of reactive oxygen species leading to\nlipid membrane peroxidation. We previously reported the development\nof a new class of ferroptosis inducers referred to as CETZOLEs with\nCC<sub>50</sub> values in the low micromolar range. Structure–activity\nrelationship study of these compounds led to the development of more\npotent analogs with CC<sub>50</sub> values in the nanomolar range.\nCells exposed to these compounds displayed the hallmarks of ferroptosis\nincluding cell death through ROS accumulation. Cancer cells were found\nto be more sensitive to these compounds than normal cells. Proteomic\nstudies using covalent and affinity probes led to the identification\nof cystathionine β-synthase, peroxiredoxins, ADP/ATP carriers,\nand glucose dehydrogenase as enriched proteins. The binding of CETZOLEs\nto these proteins as well as GPX4 was validated by Western blotting.\nThis group of proteins is known to be associated with cellular antioxidant\npathways.