# A tangible method to assess native ferroptosis suppressor activity

## TL;DR

The paper introduces a new method to measure the activity of proteins that prevent ferroptosis, a type of cell death linked to various diseases.

## Contribution

A tangible method using affinity-purified GPX4 and FSP1 to assess native ferroptosis suppressor activity is introduced.

## Key findings

- The method allows for measuring native GPX4 activity and evaluating its inhibitors like RSL3.
- WIN62577 is identified as a specific inhibitor of ferroptosis suppressor protein 1 (FSP1).
- The approach overcomes limitations of conventional GPX4 activity assays using whole-cell lysates.

## Abstract

Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.

•A versatile method for assaying activity of the ferroptosis guardians GPX4 and FSP1•The affinity-purified protein method captures a snapshot of native activity•GPX4 prepared in this way responds to RSL3 inhibition•Identification of WIN62577 as a human FSP1-specific inhibitor

A versatile method for assaying activity of the ferroptosis guardians GPX4 and FSP1

The affinity-purified protein method captures a snapshot of native activity

GPX4 prepared in this way responds to RSL3 inhibition

Identification of WIN62577 as a human FSP1-specific inhibitor

To comprehend the regulatory mechanisms of ferroptosis, evaluating the enzymatic activity of GPX4, a key player in inhibiting lethal lipid peroxidation, is essential. Conventional GPX4 activity assays using whole-cell lysate have obstacles due to the presence of oxidoreductases contained in cell lysates. Additionally, challenges arise in producing recombinant GPX4 in bacteria, with the enzyme exhibiting unexpected resistance to well-known GPX4 inhibitors. To address these challenges, this study introduces a GPX4-specific activity assay employing affinity-purified GPX4 and purified lipid hydroperoxide. This method was extended to FSP1, opening avenues for investigating ferroptosis suppressor activities.

Lipid peroxidation triggers ferroptosis, a promising therapeutic target. Nakamura et al. present a tangible method that utilizes affinity-purified GPX4 and FSP1 to obtain a snapshot of native anti-ferroptotic activity. This assay opens up avenues for evaluating alternative ferroptosis regulatory mechanisms and for screening ferroptosis-inducing agents targeting key suppressors.