The ferroptosis inducing compounds RSL3 and ML162 are not direct inhibitors of GPX4 but of TXNRD1

Ferroptosis is defined as cell death triggered by iron-dependent lipid peroxidation that is preventable by antioxidant compounds such as ferrostatin-1. Endogenous suppressors of ferroptosis include FSP-1 and the selenoprotein GPX4, the latter of which directly enzymatically reduces lipid hydroperoxides. Small molecules that trigger ferroptosis include RSL3, ML162, and ML210; these compounds are often used in studies of ferroptosis and are generally considered as GPX4 inhibitors. Here, we found that RSL3 and ML162 completely lack capacity of inhibiting the enzymatic activity of recombinant selenoprotein GPX4. Surprisingly, these compounds were instead found to be efficient inhibitors of another selenoprotein, TXNRD1. Other known inhibitors of TXNRD1, including auranofin, TRi-1 and TRi-2, are also efficient inducers of cell death but that cell death could not be suppressed with ferrostatin-1. Our results collectively suggest that prior studies using RSL3 and ML162 may need to be reevaluated in the context of ferroptosis with regards to additional enzyme targets and mechanisms of action that may be involved.

Automated summary

Ferroptosis refers to cell death triggered by iron-dependent lipid peroxidation, which can be prevented by antioxidant compounds like ferrostatin-1. Endogenous suppressors of ferroptosis include FSP-1 and the selenoprotein GPX4, while small molecules like RSL3 and ML162 can induce ferroptosis by inhibiting GPX4. However, our findings suggest that RSL3 and ML162 may not inhibit GPX4 enzymatic activity as previously believed, but instead function as inhibitors of another selenoprotein, TXNRD1. These results highlight the need for reevaluation of previous studies using RSL3 and ML162 in the context of ferroptosis.