Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models

Abstract

Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.

Figure 1

The effects of ferrostatins in Huntington’s disease (HD), periventricular leukomalacia (PVL), and kidney proximal tubules cell death models. (a) Chemical structure of ferrostatins. (b) Effect of ferrostatins on cell survival in an HD brain-slice model. YFP = yellow fluorescent protein transfection control. httN90Q73 is mutant huntingtin (N-terminal 90aa with a Q73 repeat). KW + SP is a combination used as a positive control for protection. (c) Dose–response test of the effect of ferrostatins in a model of PVL. Cys = Cystine supplementation, a positive control for cell death rescue. (d) Effect of ferrostatins, at various concentrations, in a primary kidney renal tubule damage model.

Figure 2

Fer-1 inhibits the oxidative destruction of unsaturated fatty acids. (a) Significant (P < 0.05) changes in metabolite levels in HT-1080 cells treated with erastin (10 μM, 6 h) versus DMSO (left) or with erastin + Fer-1 (1 μM) versus erastin alone (right). 2-LG, 2-linoleoylglycerol; 1-LG, 1-linoleoylglycerol; 2-AG, 2-arachidonoyl glycerol. (b) Spot dilutions of Saccharomyces cerevisiaecoq3Δ cells treated with linolenic acid (LA, 500 μM) ± trolox (50 μM, a positive control antioxidant) or ferrostatin-1 (Fer-1, 10 μM).

Figure 3

Fer-1 does not inhibit all forms of ROS production or ROS-induced death. (a) Mitochondrial ROS production in response to rotenone (Rot, 250 nM, 3 h) ± Fer-1 (1 μM) was detected using MitoSOX. (b) Cardiolipin peroxidation in response to staurosporine (STS,100 nM, 3 h) detected using 10-nonyl acridine orange (NAO). Data in (a) and (b) were analyzed by one-way ANOVA ***P < 0.001, ns = not significant; (c) Lysosomal membrane permeabilization detected in response to H₂O₂ using acridine orange (AO) relocalization. An iron chelator, ciclopirox olamine (CPX), protects from lysosomal rupture.

Figure 4

SAR study of Fer-1. (a) General scheme for the synthesis of ferrostatins. (b) Fer-1 as a reducing agent: release of 2 protons and 2 electrons results in a formation of ethyl 4-(cyclohexylimino)-3-iminocyclohexa-1,5-dienecarboxylate intermediate B.

Figure 5

SAR study of Fer-1. (a) EC50, cLogP, and % DPPH inhibition of selected potent ferrostatins. DPPH: 2,2-diphenyl-1-picrylhydrazyl radical. (b) Xray structure of the most potent Fer-1 analogue (SRS11-92).