Thermal Shift Assay in Ferroptosis

Abstract

Ferroptosis is a recently described process of cell death that is dependent on unregulated cellular iron accumulation with induction of oxidative stress. Ferroptosis has been linked to several human diseases; therefore, investigations aimed at better understanding the pathway and elucidating avenues for future drug development are warranted. Current assays that target ferroptosis/oxidative stress in cells is limited to western blotting and imaging techniques, and unfortunately provide only a broad understanding that is insufficient to effectively assess novel drugs (ligands). Specifically, these assays do not provide insights about ligand interactions with specific proteins associated with these processes. Herein, we discuss a cell-based thermal shift assay that enables screening of ligands under specific cellular conditions for targeting ferroptosis and/or oxidative stress pathways. These data would provide detailed preliminary evidence required for drug development that targets this pathway.

Keywords: Cells; Ferroptosis; Iron; Oxidative stress; Thermal shift assay.

Fig. 1

Method (step-wise) of cellular thermal shift assay (CETSA). The illustration was created by BioRender.com accessed on April 1, 2023

Fig. 2

Fig. 4D used with permission from Acta Pharm Sin B. 12(5). Luo P, Liu D, Zhang Q, Yang F, Wong YK, Xia F, Zhang J, Chen J, Tian Y, Yang C, Dai L, Shen HM, Wang J. Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1. 2300–2314. Copyright Elsevier (2022) [31] Western blot showing expression of several proteins with celastrol (20 mmol/L) or DMSO treatment to LX-2 cells with increasing temperature. Proteins like PRDX1, PRDX2, PRDX4, PRDX6, and HO-1 all displayed significant thermal stabilization (higher expression, compared to control at high temperature) in the celastrol treatment group