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

Article abstract of DOI:10.1021/ja411006a

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.

Full text of DOI:10.1021/ja411006a

Article
pubs.acs.org/JACS
Open Access on 03/04/2015
Ferrostatins Inhibit Oxidative Lipid Damage and Cell Death in
Diverse Disease Models
Rachid Skouta,† Scott J. Dixon,† Jianlin Wang,§ Denise E. Dunn,∥ Marina Orman,† Kenichi Shimada,†
Paul A. Rosenberg,§ Donald C. Lo,∥ Joel M. Weinberg,⊥ Andreas Linkermann,#
and Brent R. Stockwell*,†,‡,⊗,¶
†Department of Biological Sciences, Columbia University, 550 West 120th Street Northwest Corner Building, MC 4846 New York,
New York 10027, United States
‡Department of Chemistry, Columbia University, New York, New York 10027, United States
§Department of Neurology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States
∥Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27704,
United States
⊥Department of Internal Medicine/Nephrology, University of Michigan, Ann Arbor, Michigan 48109, United States
#Clinic for Nephrology and Hypertension, Christian-Albrechts-University, Kiel 24105, Germany
⊗Department of Systems Biology and ¶Howard Hughes Medical Institute, Columbia University, New York, New York 10027, United
States
S
* Supporting Information
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.
act to catalyze free radical formation directly in the cytosol (e.g.,
via Fenton chemistry) and/or as a cofactor for an enzyme
essential for the ROS production that precedes cell death.8
Here, we provide evidence that Fer-1 (Figure 1a) is a drug-
like, easily synthesized, potent inhibitor of ferroptosis that acts
via a reductive mechanism to prevent damage to membrane
lipids and thereby inhibit cell death.
Fer-1 is an arylalkylamine. It is established that diarylamines
and hindered dialkylamines are antioxidants (also known as
scavengers or reducing agents) in the food industry to prevent
spoilage,9 and in the materials industry to slow the oxidative
degradation of polymers, plastics, and fine chemicals.10 In the
chemical industry, these antioxidants retard autoxidation of
substrates, such as rubbers and plastics. The autoxidation is
caused primarily by radical chain reactions between oxygen and
the substrates. To prevent autoxidation form occurring,
hindered amines such as 2,2,6,6-tetramethyl-piperidine, for
example, have been used in the synthesis of highly stable
polyvinylchlorides,11 plastics, and rubbers.12 In an academic
setting, Sheltons,13−15 and Ingold16−18 were among the first to
report in the 1960s studies on the inhibition of free radical
INTRODUCTION
■
Reactive oxygen species (ROS) are required for a wide range of
normal biological processes,1 but also have been linked to tissue
degeneration2 and cell death.3 Thus, there is a clear rationale
for the development of small molecule antioxidants with high
potency and specificity for damage-inducing or death-
promoting ROS.4 Ideally, such compounds would be readily
synthesized and selectively prevent the formation or accumu-
lation of ROS that are damaging or lethal to cells, without
impinging upon the production of normal homeostatic ROS.
We recently identified ferrostatin-1 (Fer-1) as a potent and
selective inhibitor of ferroptosis, a form of nonapoptotic, iron-
dependent, oxidative cell death.4 Ferroptosis can be triggered in
cancer cells by several small molecules, including erastin,
sulfasalazine, and RSL3, that perturb redox homeostasis,
allowing the iron-dependent accumulation of lethal ROS.4−7
In human HT-1080 fibrosarcoma cells, erastin treatment blocks
the uptake of cystine, the oxidized form of cysteine,4 which is
the rate-limiting precursor for the production of the antioxidant
tripeptide glutathione. Erastin treatment leads after several
hours to the accumulation of lipid ROS, as detected using the
oxidizable lipid ROS probe C11-BODIPY, and, ultimately, cell
death.4 Both C11-BODIPY oxidation and cell death are blocked
by Fer-1.4 The role of iron in ferroptosis is unclear, and it may
Received: November 6, 2013
Published: March 4, 2014
© 2014 American Chemical Society
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chain mechanisms of autoxidation of organic materials. Since
then, there has been a tremendous interest in investigating the
effect of phenols, diphenylamines, and hindered alkylamines as
potential antioxidants in medically relevant systems in the
search for therapeutics.19,20 Recently, Michelle Coote and co-
workers reported21 ab initio calculations on a new mode of the
inhibition of hindered amine light stabilizers (HALSs). These
amines are based on radical-trapping antioxidants (RTAs).
Secondary amines, in particular diarylamines, are regarded as
important scavengers that prevent radical chain reactions and
that can serve as antioxidants, since their reducing activity allow
them to act as radical scavengers.22,23 Most studies of amine
antioxidants have been focused either on diarylamines or
hindered dialkylamines. In this study, we report a third family
of this type of antioxidant, the arylalkyl amines, exemplified by
Fer-1, and its therapeutic relevance.
RESULTS AND DISCUSSION
■
We previously showed that Fer-1 attenuates oxidative, iron-
dependent cell death in cancer cells treated with small
molecules that block cystine import and glutathione
production, as well as cell death in rat organotypic hippocampal
brain slices treated with toxic concentrations of glutamate,4
which also blocks cystine import.24,25 We hypothesized that
Fer-1 would be effective at preventing other forms of cell death
involving iron-dependent oxidative stress and/or glutamate
toxicity. Huntington’s disease (HD) is reported to involve
perturbation of glutamate, glutathione, and iron levels;26 the
accumulation of lipid oxidation breakdown products;27 and
nonapoptotic neuronal cell death.28 We therefore tested
whether Fer-1 could prevent, in rat corticostriatal brain slices,
cell death induced by the expression, via biolistic transfection,
of a huntingtin (htt) exon 1 fragment with a pathogenic repeat
(73Q) (mN90Q73), along with yellow fluorescent protein
(YFP) to mark transfected neurons29 (Figure 1b). Slices were
treated with DMSO (vehicle control), a positive control death
inhibitor combination of the adenosine A2A receptor
antagonist KW-6002 (KW, 50 μM) and the JNK inhibitor
SP600125 (SP, 30 μM), or ferrostatins (Fer-1 or SRS11-92) at
increasing concentrations (1 nM to 1 μM). Four days later, the
number of healthy medium spiny neurons (MSNs) was
quantified, as previously described.29 A significant increase in
the number of healthy MSNs was observed upon Fer-1 and
SRS11-92 treatment at 10 nM, 100 nM, and 1 μM. Moreover,
with 1 μM treatment of Fer-1, the number of healthy MSNs
was statistically indistinguishable (P > 0.05) from both the YFP
(no htt) control and the control inhibitor combination (KW +
SP) (Figure 1b).
We then tested the effect of Fer-1 in an in vitro model of
periventricular leukomalacia (PVL), a syndrome afflicting
infants born prematurely that is caused primarily by the death
of developing oligodendrocytes (OLs).30 It has been suggested
that OL death is iron-dependent; in autopsy studies, elevated
levels of lipid ROS biomarkers are observed,31−34 potentially
suggesting the involvement of ferroptosis. To trigger death,
cultured OLs were exposed to cystine-free conditions, which
ultimately deplete glutathione. Fer-1, SRS11-92 (Figure 1c),
and Fer-1 analogues (Supporting Information Figure S1) fully
protected OLs from cystine deprivation when tested at 100 nM
(Figure 1c).
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.
elements of rhabdomyolysis-induced acute kidney injury.35 Cell
death in this model is known to involve oxidative lipid
damage;35 we therefore suspected that Fer-1 might be
protective. Hydroxyquinoline and ferrous ammonium sulfate
(HQ + Fe; 10 μM each) were used to induce cell death, which
was quantified by monitoring LDH release after 60 min. In this
model, Fer-1, SRS11-92, and analogues prevented lethality
(Figure 1d, Supporting Information Figure S2). Together, the
results of these assays demonstrated that Fer-1, SRS11-92, and
analogues are potent inhibitors of a variety of clinically relevant
oxidative cell death phenotypes. By implication, cell death in
these diverse models may involve, at least in part, the induction
of ferroptosis.
Using the general lipid ROS probe C11-BODIPY (581/
591),36 we previously suggested that erastin-induced ferroptosis
involves Fer-1-sensitive oxidative lipid modification.4 We
confirmed this result using an independent assay. HT-1080
cells were incubated with an alkyne containing form of the
oxidizable fatty acid linoleic acid (linoleamide alkyne, LAA) and
treated with erastin (10 μM) +/− Fer-1 or SRS11-92 (both at 1
μM). After 7.5 h, cells were washed, fixed, and permeabilized,
and a copper-catalyzed cycloaddition reaction (“click” reaction)
was performed with fluorescein azide. When viewed under a
microscope, cells treated with erastin displayed accumulation of
fluorescein-positive puncta, indicative of the formation of
oxidative breakdown products of LAA, while co-treatment with
Fer-1 or SRS11-92 prevented the accumulation of this marker
(Supporting Information Figure S4).
The above results suggested that the oxidative destruction of
specific fatty acids was likely to occur during ferroptosis. To
search in an unbiased manner for Fer-1-sensitive effects on
specific membrane lipids or other metabolites, we characterized
changes in the metabolome of HT-1080 cells treated with
Finally, we tested the effects of Fer-1, SRS11-92, and selected
analogues in a model of iron-induced cell death in freshly
isolated mouse kidney proximal tubules that simulates major
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erastin (10 μM) +/− Fer-1 (1 μM) (Figure 2a). Cells from
four independent biological replicates were harvested following
(PUFAs) and PUFA-derivatives, including eicosapentaenoate
(20:5n3), linoleate (18:2n6), linolenate (18:3n3/6), and
docosahexaenoate (22:6n3). PUFAs and PUFA-containing
lipids are highly susceptible to oxidation, via both enzymatic
(e.g., lipoxygenase-enzyme-mediated) and non-enzymatic (e.g.
iron-dependent Fenton chemistry-mediated) processes.38
A
parsimonious explanation of these results is that under
conditions that favor ferroptosis (e.g., cystine and glutathione
depletion) specific membrane PUFAs are oxidized and
subsequently fragmented, resulting in the net depletion of
these species, as detected in our MS analyses. Fer-1 is able to
prevent the depletion of these species by inhibiting their
oxidative destruction. Mechanistically, we hypothesize that Fer-
1 acts either to reduce a lipid peroxide to an alcohol (R-OOH
→ R-OH) and/or to intercept and scavenge a lipid radical
through hydrogen atom transfer or direct reduction (R-O· →
R-OH). Both mechanisms would explain the sparing of PUFAs
by Fer-1 under ferroptosis-inducing conditions. While these
results cannot conclusively rule out the possibility that the
observed Fer-1-sensitive changes in lipid metabolites are merely
a correlate of rescue from erastin-induced death by another
means, we speculate that the oxidative destruction of these
lipids contributes directly to cell death, either through the
physical disruption of the plasma membrane permeability
barrier or through the formation of PUFA breakdown products
(e.g., reactive aldehydes) that are toxic to cells.
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 cerevisiae coq3Δ cells
treated with linolenic acid (LA, 500 μM) trolox (50 μM, a positive
control antioxidant) or ferrostatin-1 (Fer-1, 10 μM).
On the basis of these results, we hypothesized that the Fer-1-
sensitive oxidative destruction of PUFAs is sufficient for
ferroptotic cell death. This hypothesis is difficult to test in
mammalian cells, given the compositional complexity of the
plasma membrane. Thus, we employed a yeast (S. cerevisiae)
model of cell death, where it is possible to isolate the lethal
effects of individual PUFAs.39 PUFAs are not normally present
in the plasma membrane of S. cerevisiae, but these cells normally
tolerate growth in the presence of high concentrations of
PUFAs (which are susceptible to auto-oxidation), in part,
through a moonlighting antioxidant function of the mitochon-
drial electron carrier coenzyme Q.40 S. cerevisiae cells in which
coenzyme Q biosynthesis has been eliminated by deletion of
the gene COQ3 (coq3Δ) are hypersensitive to incubation in
PUFAs, providing a model of PUFA-oxidation-induced death.
Thus, coq3Δ cells were incubated with 500 μM linolenic acid
(LA, 18:3) for 6 h +/− Fer-1 (10 μM) or a positive control
lipophilic antioxidant (trolox, 50 μM), then spotted in a
dilution series on agar to examine clonogenic growth. Like
trolox, Fer-1 restored the clonogenic growth of LA-treated
coq3Δ cells. Thus, Fer-1 can inhibit the loss of clonogenic
growth potential induced by PUFA auto-oxidation, entirely
consistent with a model where Fer-1 prevents cell death by
inhibiting the oxidative destruction of membrane lipid PUFAs.
To obtain further insight into the Fer-1 protective
mechanism, we designed a three-step synthesis based on the
previously reported synthetic route of Fer-1.4 A nucleophilic
aromatic substitution reaction (SNAr) between the commer-
cially available ethyl 4-chloro-3-nitrobenzoate and cyclohexyl-
amine followed by catalytic hydrogenolysis of the nitro group
provided the desired Fer-1 derivatives.40 The aniline of the
latter compounds was reacted through reductive amination in
the presence of sodium triacetoxyborohydride or alkylation
conditions in the presence of either arylaldehydes41 or 1-
(bromomethyl)aryl compounds, respectively (Figure 4a). We
used this general route to synthesize 67 analogues. We
established a structure−activity relationship (SAR) for the
6 h of compound treatment, before death became apparent, and
subjected to GC/MS and LC/MS/MS analysis. From a set of
328 metabolites consistently identified in all conditions, 78
were altered significantly (either depleted or enriched) in
erastin versus DMSO treatment conditions (P < 0.05, Figure
2a, Supporting Information Table S1). Consistent with our
proposed mechanism of action for erastin as an inhibitor of
cystine import via the system xc− transporter,4 we detected
significant erastin-induced depletion of cysteine and cysteinyl-
glycine (indicated in Figure 2a). We likewise observed
substantial depletion of glutathione, suggesting that the
synthesis of this tripeptide is compromised in these cells
when cystine import is inhibited, as expected. Furthermore, the
most highly enriched metabolite was the glutathione analogue
ophthalmate (12-fold increase in erastin versus DMSO-treated
control), which has a methyl group in place of the thiol found
in glutathione. Ophthalmate accumulation has previously been
observed under conditions where glutathione, and presumably
cysteine, are depleted.37 Under these conditions, the enzymes
of the glutathione synthesis pathway use 2-aminobutyrate in
place of cysteine to form ophthalmate. While this compound is
redox inactive, it may serve to inhibit GSH-consuming
enzymes, thereby conserving intracellular GSH stores.37
Ophthalmate is thus a potential metabolic biomarker for
cysteine-depleted cells that could be used to assess the efficacy
of treatments that target cystine import or cysteine
metabolism.37 The finding that the levels of ophthalmate are
elevated following erastin treatment is consistent with our
model of erastin’s mechanism of action.
Among the metabolites affected by erastin treatment, the
levels of small subset (17/78 = 22%) were observed to be
significantly (P < 0.05) closer to baseline (i.e., maintained at
normal levels) when cells were co-treated with Fer-1. While
Fer-1 co-treatment did not significantly change the observed
levels of cysteine, glutathione, or ophthalmate (as expected), it
did preserve the normal levels of polyunsaturated fatty acids
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To test this hypothesis, we evaluated the 67 analogues for
their ability to prevent erastin-(10 μM)-induced cell death4−6
(see Supplementary Synthesis Section in Supporting Informa-
tion). Cell death inhibition (EC50) values were determined by
treating HT-1080 fibrosarcoma cells with a lethal concentration
of erastin (10 μM) in the presence of each ferrostatin analogue
in a 10-point, 2-fold dilution series starting at 4 μg/mL, or a 20-
point, 2-fold dilution starting at 2 μg/mL, for subsequently
identified more potent compounds. These results are presented
in Tables S2−S8 in the Supporting Information.
To assess the importance of the primary aromatic amine in
the activity of ferrostatins, we designed mono- and
disubstituted amines (Supporting Information Tables S5−S7).
We hypothesized that if the above mechanism were correct;
these modifications should decrease or abolish the ability of the
compounds to prevent erastin-induced ferroptosis. In these
studies, the primary aromatic amine of the ferrostatin
compounds was used as a starting material for a reductive
amination or alkylation reaction using arylaldehydes or 1-
(bromomethyl)aryl compounds, respectively (Supporting In-
formation Tables S5−S7). In addition, other electrophiles, such
as acylchlorides, alkylchloroformates, or benzylchloroformates,
were also used to generate ferrostatin derivatives efficiently
(Supporting Information Table S5).
Figure 3. Fer-1 does not inhibit all forms of ROS production or ROS-
induced death. (a) Mitochondrial ROS production in response to
We observed that, first, all analogues with a nitro group in
place of the amine were inactive (Supporting Information
Table S8, entry 1 vs entry 2), confirming the concept that this
amine is essential for suppression of ferroptosis. In addition,
analogues lacking a hydrophobic cyclohexylamine moiety were
not active (Supporting Information Table S8, entry 1 vs entries
3 and 4). Second, introduction of heteroatoms into the N-
cyclohexyl moiety resulted in consistent reductions in potency,
consistent with the hypothesis that the hydrophobicity of this
portion of the scaffold is crucial for anchoring within lipophilic
membrane environments. Third, modifications of the ethyl
ester4 (Supporting Information Table S4 and Scheme S1),
involving substantial extensions, were generally well-tolerated,
and may serve as substrates for future probes. Fourth, both
amines are essential for full activity; while there was no
difference in potency between the nonalkylated and mono-
methylated analogues (Supporting Information Table S5, entry
6 vs entry 7), the N,N-dimethyl substituted derivative was 43-
fold less potent than the mono-N-methyl substituted derivative
(Supporting Information Table S5, entry 7 vs entry 8). Thus, it
appears that the ability to oxidize both amines is crucial for the
potency of ferrostatins (Figure 4b).
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.
In addition, compounds with electron withdrawing groups,
such as methyl-, benzyl-, or tert-butylcarbamates, as well as
amides, block tautomerization, which results in the inhibition of
the release of 2 protons and 2 electrons (Figure 4b). This
mechanism was supported by the loss of the activity of these
analogues (Supporting Information Table S5, entries 2−5).
Indeed, a tertiary amine (dioctyl) derivative was 190-fold less
active than a secondary amine (monooctyl) (Supporting
Information Table S5, entry 9 vs entry 10). These data support
our hypothesis that the activity of ferrostatins requires both
secondary amines (Figure 4b). Fifth, we extended the reductive
amination or the selective monoalkylation reaction of Fer-1 to
aryl aldehydes and aryl halides, respectively (Supporting
Information Tables S6 and S7). These aryl aldehydes and
aryl halides were ortho, meta, or para mono- or disubstituted
with electron withdrawing or electron donating groups (−Cl,
−Br, −F, −CN, −CF3, −NO2, esters, or −OCH3). In addition,
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.
Fer-1 scaffold (Figure 4a, Tables S2−S8 and Schemes S1−S5).
We sought to test the hypothesis that Fer-1 acts as an
antioxidant.4 Examination of the Fer-1 structure suggested a
possible release of two protons and two electrons, through the
tautomer intermediate compound A, resulting in the formation
of a redox-stable compound B (Figure 4b). This mechanism
could form the basis for the antioxidant activity of Fer-1.
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we also incorporated a nitrogen atom in the phenyl ring in the
design of the pyridine and pyrimidine analogues of Fer-1
(Supporting Information Table S7). Overall, the introduction
of these variations to the mono-alkylated analogues did not
affect potency, suggesting that these modifications did not
inhibit the electron delocalization and release of reducing
equivalents into the cell. These analogues further support our
hypothesized model of Fer-1’s antioxidant activity. Sixth and
finally, we modified the aniline ring of the parent compound
(Fer-1) and created additional Fer-1 analogues with (1) a
nitrogen incorporated in the aniline ring (pyridine) (Support-
ing Information Scheme S2); and (2) with various substitutions
(F, Cl, CH3) in the aniline ring (Supporting Information
Schemes S3−S5). Overall, we observed a loss of potency with
the pyridine moiety analogues (Supporting Information
Scheme S2), while the aniline-substituted analogues have
equivalent or better potency than the Fer-1 (Supporting
Information Schemes S3−S5). Overall, this series of Fer-1
analogues provided insight into the Fer-1 mechanism of action
and also allowed us to identify analogues with greater potency
and improved properties. Indeed, we discovered a number of
compounds (SRS11-92, SRS12-45, SRS13-35, and SRS13-37)
that were more potent than Fer-1 and may be suitable for
further translational studies (Figure 5a). For example, SRS11-
analogues scavenged the DPPH radical by 60−90% within 30
min (Figure 5a). Consistent with the notion that the lipophilic
character of the N-cyclohexyl ring is essential for cell death
inhibition in cells, but not intrinsic antioxidant activity,4 Fer-1
analogues bearing heteroatoms substitutions such as O- or N-
methyl in this moiety retained antioxidant activity in the DPPH
assay (68% and 86% reduction of DPPH, respectively,
Supporting Information Figure S3), despite resulting in much
lower potency in the HT-1080 death-suppression assay.
Conversely, analogues with electron-withdrawing functional
groups on the primary amine did not scavenge the DPPH
radical (0−10%, Supporting Information Figure S3) and did
not prevent cell death, suggesting that these groups act to
prevent the delocalization and oxidation necessary for radical
scavenging (Figure 4b).
CONCLUSION
■
In conclusion, the testing of ferrostatins, generated via an
efficient three-step synthesis, has provided insights into the
ferroptotic phenotype, confirming that it involves the depletion
of specific membrane lipids, most likely due to oxidative
destruction. As Fer-1 and analogues are protective against cell
death in a brain slice model of HD, an oligodendrocyte model
of PVL, and in isolated kidney proximal tubules that model
kidney dysfunction, these results imply that these processes
may converge upon or require oxidative destruction of specific
PUFA-containing membrane lipids and might represent
examples of ferroptotic cell death that occur during pathological
situations. The ferrostatins represents useful probes with which
to dissect ferroptosis in a variety of contexts and could form the
basis of future drugs to combat lipid-peroxidation-mediated
tissue injury in diverse diseases.
ASSOCIATED CONTENT
* Supporting Information
Additional synthetic and characterization details. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
bstockwell@columbia.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was funded by grants from the New York State Stem
Cell Science (NYSTEM, Contract No. C026715 for the CPS
Facility) and the US National Institute of Health (NIH
R01CA097061, R01GM085081, R01 CA161061, HD18655,
NS066019, and DK34275), the Whitehall Foundation, the
William Randolph Hearst Foundation, Alzheimer's Drug
Discovery Foundation, and the Baby Alex Foundation. B.R.S.
is an Early Career Scientist of the Howard Hughes Medical
Institute. S.J.D. was supported by a K99 Award from the
National Cancer Institute (1K99CA166517-01). We acknowl-
edge the assistance of J. Decatur, Y. Itagaki, G. Parkin, and J.
Palmer, and NSF grants CHE 0840451 and CHE-06196, and
NIH grant 1S10RR025431-01A1.
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).
92 was 15-fold more potent than the parent Fer-1, with an EC50
= 6 nM (Figure 5a, entry 3). An X-ray structure of SRS11-92
analogue was obtained (Figure 5b) to confirm its purity and the
orientation of both secondary amines.
In parallel with the above cell-based assays, we used a 2,2-
diphenyl-1-picrylhydrazyl (DPPH) reduction assay4,42−44 to
examine the ability of each analogue to reduce this free radical
in an in vitro test of intrinsic reducing (and hence antioxidant)
capacity. The reduction of the DPPH radical by ferrostatins
results in a decrease in absorbance, indicating the radical
quenching capacity of the ferrostatins. Antioxidant active
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