Reactivity-based probe of the iron(II)-dependent interactome identifies new cellular modulators of ferroptosis

Article abstract of DOI:10.1021/jacs.0c06709

Ferroptosis is an iron-dependent form of cell death resulting from loss or inhibition of cellular machinery that protects from the accumulation of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing function in normal cellular homeostasis, but certain cancers exploit and become highly dependent on specific nodes of the pathway, presumably to survive under conditions of increased oxidative stress and elevated labile ferrous iron levels. Here we introduce Ferroptosis Inducing Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe that couples Fenton-type reaction with ferrous iron to subsequent protein labeling via concomitant carbon-centered radical generation. We show that FIPC-1 induces ferroptosis in susceptible cell types and labels cellular proteins in an iron-dependent fashion. Use of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD) superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and uncovers new targets for the therapeutic modulation of ferroptosis.

Full text of DOI:10.1021/jacs.0c06709

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Article
Reactivity-Based Probe of the Iron(II)-Dependent Interactome
Identifies New Cellular Modulators of Ferroptosis
Ying-Chu Chen, Juan A. Oses-Prieto, Lauren E. Pope, Alma L. Burlingame, Scott J. Dixon,
and Adam R. Renslo*
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ABSTRACT: Ferroptosis is an iron-dependent form of cell death
resulting from loss or inhibition of cellular machinery that protects
from the accumulation of lipid hydroperoxides. Ferroptosis likely
serves a tumor suppressing function in normal cellular homeostasis,
but certain cancers exploit and become highly dependent on
specific nodes of the pathway, presumably to survive under
conditions of increased oxidative stress and elevated labile ferrous
iron levels. Here we introduce Ferroptosis Inducing Peroxide for
Chemoproteomics-1 (FIPC-1), a reactivity-based probe that
couples Fenton-type reaction with ferrous iron to subsequent
protein labeling via concomitant carbon-centered radical gener-
ation. We show that FIPC-1 induces ferroptosis in susceptible cell
types and labels cellular proteins in an iron-dependent fashion. Use
of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and
protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the
PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD)
superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known
ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly
synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and
uncovers new targets for the therapeutic modulation of ferroptosis.
subtype of tumor-killing cells, drives ferroptosis in tumor cells,
consistent with a tumor-suppressor function of the pathway,
suggesting that its activation in certain tumors may have
therapeutic potential. Moreover, so-called drug tolerant
“persister” cells that survive targeted kinase inhibitor therapy
and from which truly resistant tumor cells likely emerge were
found to be highly reliant on GPX4 activity for survival and
sensitive to ferroptosis.18 Thus, there is considerable interest in
the potential of ferroptosis modulating small molecules as
therapeutics.
Multiple classes of ferroptosis inducing small molecules have
been described to date. Thus, erastin and sorafenib block
cystine uptake through inhibition of the system Xc−, while
(1S,3R)-RSL3 and its analogues inactivate GPX4.2 The small
molecule FIN56 acts by depleting the GPX4 protein and
INTRODUCTION
■
Ferroptosis is an iron-dependent form of nonapoptotic cell
death resulting from the accumulation of lipid hydroperoxides
due to the loss or inhibition of the cellular machinery that
make up a lipid peroxide defense network.1−4 Three key nodes
of this network have been identified: the glutathione (GSH)-
dependent lipid peroxidase GPX4,5−7 the cystine−glutamate
antiporter system Xc−,8−10 and most recently a GSH-
independent pathway involving the lipophilic antioxidant
coenzyme Q10 (CoQ10) and the CoQ oxidoreductase
ferroptosis suppressor protein 1 (FSP1).11,12 Ferroptosis can
be inhibited by iron chelators like desferoxamine (DFO) and
small molecule antioxidants such as ferrostatin-113,14 (Fer-1),
which prevent lipid reactive oxygen species accumulation.
Executioner caspases are not significantly activated in
ferroptosis, and thus caspase inhibition does not protect cells
from ferroptosis. Like apoptosis, however, ferroptosis is
thought to serve a tumor-suppressive function,15,16 tipping
oxidatively stressed cells toward death.
Received: June 22, 2020
Ferroptosis has been implicated in various disease states,
including neurodegeneration, ischemic organ injury, and
cancer.3 A recent study17 revealed that CD8+ T cells, a classic
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simultaneously causing depletion of CoQ10.19 Perhaps most
enigmatic among known ferroptosis inducers is FINO2 (Figure
1), a 1,2-dioxolane first described by Woerpel and co-
To explore this possibility, we designed and synthesized the
probe FIPC-1 for Ferroptosis Inducing Peroxide for Chemo-
proteomics 1 (Figure 1). Structurally, FIPC-1 is inspired by
FINO2 but is further endowed with a protein cross-linking
functionality borrowed from the well-studied antimalarial
arterolane.22 The adamantane-2,3′-[1,2,4]-trioxolane ring
system present in FIPC-1 is known to react selectively with
Fe2+ over other divalent metal ions, with initial O−O bond
scission followed rapidly by β-scission to form a carbon-
centered radical intermediate capable of protein cross-linking
(Figure 1).23 The Fe2+ promoted reactivity of 1,2-dioxolanes,
by contrast, involves sequential one-electron reductions to
afford diols,24 making dioxolanes like FINO2 unlikely to
undergo the cross-linking reactions desirable for chemo-
proteomic applications (Figure S1). The conjoining in FIPC-
1 of Fe2+ reactivity with protein labeling obviates the need for
extra electrophilic or photoactivatable moieties; only the
addition of an alkyne is required to enable subsequent
visualization or capture of protein adducts for analysis.
Using analogues that bridge the structural differences
between FINO2 and FIPC-1, we demonstrate that FIPC-1
both induces ferroptosis and forms protein adducts in an iron-
dependent fashion, as expected. Using FIPC-1 in a quantitative
chemoproteomics workflow, we observed reproducible enrich-
ment of ∼45 proteins, including multiple oxidoreductases and
several proteins in the thioredoxin network, both protein
families having been previously linked to ferroptosis.25 Of
greatest interest, however, were a small number of targets that
showed “saturable” modification by FIPC-1 in competitive
tandem-mass tag (TMT) labeling experiments. These included
the oxidoreductases AKR1B1 and CBR1, two protein disulfide
Figure 1. Structures of ferroptosis inducing peroxides FINO2 and
FINO3 (top) and putative mechanism of FIPC-1 mediated protein
labeling (bottom).
workers.20 In a subsequent report exploring possible
mechanism(s) of FINO2 action, the compound was found
not to alter glutathione homeostasis, nor to inhibit GPX4
directly.21 FINO2 was shown to oxidize ferrous iron to the
ferric state, consistent with a role for the peroxide bond in an
initial Fenton-type reaction with labile ferrous iron. However,
the endoperoxide function, while necessary, is not alone
sufficient to induce ferroptosis. Thus, FINO2 analogues with
seemingly minor structural modification, such as one carbon
homologation of the hydroxy ethyl side chain, were without
significant activity.21 This suggested to us that rather than
conferring a generic peroxidic insult, FINO2 likely interacts
with specific macromolecules, possibly including new, as yet
unappreciated modulators of ferroptosis.
Figure 2. Trioxolane 3 is a peroxide-dependent inducer of ferroptosis. (A) Structures of 3 and nonperoxidic control 4. (B) Viability at 24 h of HT-
1080 cells cotreated with 3 (40 μM) and increasing concentrations of ferrostatin-1, necrostatin-1, or z-VAD-FMK. (C) Viability at 24 h of HT-
1080 cells cotreated with a cytotoxic dose of 3 (40 μM) or DMSO and increasing concentrations of the iron chelator DFO. (D) Lipid peroxidation
in HT-1080 cells assessed by flow cytometry using C11 BODIPY after cells were incubated with vehicle (DMSO) or ferroptosis inducers (10 μM)
with or without DFO (50 μM) for 5 h. (E) Compound 3 (20 μM) but not control 4 induced lipid peroxidation as detected with C11 BODIPY
581/591 in HT-1080PM‑mTq cells expressing mTurquoise protein in plasma membrane (green). Ferrostatin-1 (1 μM) prevented 3-induced lipid
peroxidation. Scale bar = 20 μm. (F) Lipids whose abundance was significantly altered in HT-1080 cells upon treatment for 5 h with 3. PUFA-
containing phosphatidylethanolamine ether lipids (ePE) are colored blue and triacylglycerols (TAG) orange.
B
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isomerases (PDIA6 and P4HB), the mitochondrial peroxir-
edoxin PRDX3, and most intriguingly NT5DC2, a functionally
uncharacterized member of the HAD superfamily of
phosphatases. Genetic and pharmacological modulation of
these targets identified P4HB and NT5DC2 as most closely
linked to the ferroptotic phenotype; knockdown of either
target sensitized cells to erastin and RSL3, while a known PDI
inhibitor directly induced ferroptosis and synergized with
erastin. Overall, our studies provide a new tool for chemo-
proteomic studies of the iron(II)-dependent interactome in
live cells. As applied specifically to a ferroptosis sensitive cell
type, FIPC-1 revealed new cellular modulators of ferroptosis
and possible drug targets for therapeutic intervention in cancer
and other disease states where the induction of ferroptosis is
desirable.
alone sufficient for ferroptosis induction by 1,2,4-trioxolanes,
much as is also true for FINO2 and its analogues. Moreover,
these studies confirmed that analogue 3, on which FIPC-1 is
based, is a bona fide inducer of ferroptosis.
To further validate that 3 induces lipid peroxidation, a
defining event in ferroptosis,13,26 HT-1080 cells were treated
with FINO2 or 3, and the increase in fluorescence intensity of
lipid peroxidation was evaluated with the lipid probe C11
BODIPY 581/591, as recorded by flow cytometry. Similar to
FINO2, 3 produced a clear increase in lipid peroxidation and
was suppressed by cotreatment with DFO (Figure 2D). In
addition, 3-induced lipid peroxidation occurred on internal
membranes and at the plasma membrane as determined with
whole-cell imaging of HT-1080PM‑mTq cells expressing plasma
membrane-localized mTurquoise227 and could be inhibited by
cotreatment with ferrostatin-1 (Figure 2E). No increase in lipid
peroxidation was observed for the non-peroxide analogue 4 in
the same experiment, indicating that the peroxide function in 3
is necessary for the observed phenotype. These data lend
further support for the conclusion that compound 3 induces
ferroptosis in HT-1080 cells.
We next explored the effects of compound 3 on global lipid
abundance in HT-1080 cells. Consistent with previous
observations of ferroptotic cells,28 treatment with 3 resulted
in significant depletion of numerous PUFA-containing
phospholipids, notably including many PUFA-containing
phosphatidylethanolamine ether lipids (ePEs) (Figure 2F, in
blue). Compound 3 treatment also resulted in significant
accumulation of diverse triacylglycerols, possibly because of
inhibition of one or more mitochondrial proteins leading to
disruption of β-oxidation and subsequent accumulation of free
fatty acids that are stored in TAGs (Figure 2F, in orange). The
full list of lipids analyzed are provided as a data set in the
Supporting Information.
RESULTS AND DISCUSSION
■
Previously reported structure−activity relationship (SAR)
studies21 of FINO2 (1) defined the importance of the
peroxide bond and hydroxyethyl side chain for ferroptosis
induction. We explored the effect of replacing the 1,2-
dioxolane with a 1,2,4-trioxolane ring, to afford “FINO3”
(2), and then replaced the substituted cyclohexane ring of
FINO2 with the adamantane ring that we predicted would
enable protein cross-linking activity (compound 3, Figure 2A).
The 1,3-dioxolane 4, a non-peroxidic control, was also
synthesized, as was an analogue (5) in which the hydroxyethyl
side chain of 3 is constrained as part of a cyclohexane ring
(Figure S2). These analogues served as a structural and
pharmacological bridge between FINO2 and FIPC-1, the latter
being an analogue of 3 bearing an alkyne functionality for
Cu(I)-catalyzed [3 + 2] azide−alkyne cycloaddition
(CuAAC)-mediated coupling to fluorescent dyes or avidity
(biotin) reagents.
Finally, we assessed the possible effects of 3 on established
regulators of ferroptosis. Whereas 2 μM of the system Xc−
inhibitor erastin2 effectively depleted total glutathione in A549
cells, treatment with 3 at a concentration (40 μM) that
robustly induces ferroptosis had no significant effect on
glutathione levels (Figure S5). We also compared the toxicities
of RSL3 and 3 in A375 WT and GPX4 KO cell lines18 and
found the cytotoxicity of RSL3 was significantly reduced in the
GPX4 KO line, whereas compound 3 exhibited similar cell-
killing effects whether or not GPX4 was present (Figure S5).
This suggested that GPX4 is not a direct target of 3. Finally,
because susceptibility to ferroptosis has been associated with
the RAS oncogene, we evaluated the selective lethality of
erastin, RSL3, and 3 in inducible KRAS (G12D) AK210 cells
cultured with or without doxycycline for 72 h to turn ON/
OFF the KRAS (G12D) expression. We observed that cells
expressing mutant KRAS were sensitized to erastin, RSL3, and
3 (Figure S6). Together these studies suggest compound 3,
like FINO2, induces ferroptosis by a mechanism distinct from
the canonical ferroptosis inducers erastin and RSL3 and further
implied that a chemoproteomic probe based on 3 (i.e., FIPC-
1) could have utility in discerning as yet unappreciated
modulators of ferroptosis.
We next assessed the cytotoxicity of the new peroxides
versus FINO2 control in HT-1080 fibrosarcoma cells, a
ferroptosis-sensitive cell line that is widely used in studies of
the ferroptosis pathway. We found that FINO3 (EC50 = 3.2
μM) and 3 (EC50 = 9.7 μM) induced cell death at similar
concentrations as FINO2 (EC50 = 2.5 μM) after 24 h
treatment (Figure S2). Analogues 4 and 5 by contrast were
essentially nontoxic in HT-1080 cells (EC50 > 100 μM),
suggesting the importance of the peroxide function as well as
the orientation of the hydroxyethyl side in delivering a
cytotoxic insult.
To confirm that compounds 2 and 3 confer cell death via
ferroptosis, HT-1080 cells were treated with a lethal
concentration of FINO2, 2, or 3, along with increasing
concentrations of one of three different death-suppressing
compounds. We found that cell death induced by FINO2, 2,
and 3 was reversed by the antioxidant ferrostatin-1 but not by
necroptosis or apoptosis inhibitors (Figure 2B and Figure S3).
Cell death by the compounds could also be rescued with iron
chelator DFO (Figure 2C and Figure S3B). This suggested
that certain 1,2,4-trioxolanes, like the 1,2-dioxolane FINO2,
can induce ferroptosis. Because compound 3 includes the
antimalarial pharmacophore present in arterolane and
artefenomel (Figure S4A), we also tested these compounds
in HT-1080 cells. Exhibiting EC50 values similar to 3 in HT-
1080 cells, coadministration of death-suppressing compounds
indicated that arterolane, but not artefenomel, induces
ferroptosis-like killing (Figure S4B,C). Taken together, these
studies suggested that a peroxide function is necessary, but not
Next we evaluated cell killing by FIPC-1 as well as its
predicted ability to label proteins in an iron-dependent fashion.
Although somewhat less potent than FINO2 or 3, cell killing
by FIPC-1 was reversed by DFO or ferrostatin-1, but not by
necrostatin-1 or caspase inhibitor Z-VAD-FMK (Figure
S7A,B), indicating that FIPC-1, like 3, kills cells by ferroptosis.
C
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Figure 3. Cellular targets of FIPC-1 in HT-1080 cells. (A) Labeling of cellular proteins by FIPC-1 in HT-1080 cells is concentration- and iron-
dependent (FAC = ferric ammonium citrate). Coomassie blue staining was used as a loading control. (B) Labeling by FIPC-1 is reduced in a dose-
dependent fashion by pretreatment with compound 3 but not antioxidant ferrostatin-1. (C) Congruence of proteins enriched >1.4-fold by FIPC-1
(log2 > 0.5) over DMSO control in four separate proteomic experiments. (D) Volcano plot of enriched FIPC-1 target proteins found in n ≥ 2
experiments and enriched over DMSO control by fold change >1.4 (log2 > 0.5) and P < 0.05 in HT-1080 cells. (E) Interactome network
highlighting 19 proteins enriched by FIPC-1 and known to be involved in redox processes. The complete network is provided as Supporting
Information.
To explore its protein cross-linking activity, cells were treated
with FIPC-1 at 1, 10, 50, and 100 μM for 5 h or at 100 μM for
between 2 and 8 h followed by cell lysis, CuAAC reaction with
TAMRA-azide, separation by SDS-PAGE, and in-gel fluo-
rescence scanning (Figure S7C,D). These studies revealed
time- and concentration-dependent labeling of the HT-1080
proteome and identified a concentration−time regime optimal
for further experimentation. Importantly, subsequent experi-
ments in cells preconditioned with exogenous iron (as ferric
ammonium citrate, FAC) or with DFO established that protein
labeling by FIPC-1 is an iron-dependent process, as expected
(Figure 3A). Although 1 μM ferrostain-1 is sufficient to rescue
cells from FIPC-1 induced ferroptosis (Figure S7B), a 10-fold
higher concentration had no apparent effect on FIPC-1
labeling efficiency (Figure 3B). Importantly, labeling by
FIPC-1 could be blocked with increasing concentrations of 3
as competitor (Figure 3B).
and combined analysis by LC-MS/MS analysis using TMT-
based quantification. We compared lists of target proteins
enriched >1.4-fold by FIPC-1 over DMSO control in four
separate labeling experiments and found reasonable con-
gruence between experiments, including 15 protein targets
enriched in all four experiments (Figure 3C). This was
reassuring, given that a recent report29 has questioned the
reproducibility of earlier chemoproteomic studies30−32 involv-
ing cross-linking probes based on artemisinin and arterolane.
From the four data sets, a total of 45 proteins were found to
be enriched compared to DMSO control in at least two
experiments using an enrichment cutoff of fold change >1.4
(log2 > 0.5) and P value <0.05 (Figure 3D). The use of a
relatively low threshold for enrichment was employed to
broadly assess the range of potential targets of FIPC-1, given
its novel mode of activation/cross-linking as compared to
established photoaffinity or cysteine-reactive modalities. Of the
45 targets identified by these enrichment criteria, many were
involved in the regulation of cellular redox homeostasis,
including members of the thioredoxin network, the protein
disulfide isomerase (PDI) family, and NADPH-dependent
oxidoreductases. The results of a Cytoscape with STRING33,34
analysis for this subset of the interactome is shown above
To identify the proteins that associate with FIPC-1 in an
iron-dependent manner, we applied chemoproteomics with
tandem mass tag (TMT) quantification. Hence, HT-1080 cells
were treated with DMSO or FIPC-1 for 5 h, followed by cell
lysis, CuAAC reaction with biotin-azide, enrichment with
neutravidin beads, on-bead tryptic digestion, TMT labeling,
D
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Figure 4. Quantitative chemoproteomics with FIPC-1 reveals saturable targets. (A) General workflow for chemoproteomics using FIPC-1 in the
absence or presence of increasing concentrations (5, 15, or 50 μM) of competitor 3 and tandem mass tag mass spectrometry. (B) Saturable targets
exhibit a dose-dependent decrease in FIPC-1 labeling with increasing concentrations of competitor 3. (C) List of partially competed protein targets
of FIPC-1 (from experiment with 15 μM competitor 3). (D) siRNA knockdown of NT5DC2 sensitized HT-1080 cells to RSL3 and erastin. (E)
siRNA knockdown of P4HB rendered HT-1080 cells hypersensitive to RSL3 and erastin. (F) Cell viability at 24 h of HT-1080 cells treated with
reported PDI inhibitors 35G8, PACMA31 and LOC14. (G) Cell killing by PACMA31 (at 50 nM) and erastin at three concentrations alone or
combined (green); viability of HT-1080 cells at 24 h of incubation. In (D, E), data are plotted as mean s.e.m. with n ≥ 3 biological replicates.
2way ANOVA multiple comparisons; ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, ns P > 0.5.
(Figure 3E), and the complete list of enriched targets is
provided in the Supporting Information (Figure S8). Notably,
a very small number of iron-binding or utilizing proteins were
among the enriched protein targets. This was consistent with
the expectation that FIPC-1 should be activated by an
unbound, labile ferrous iron, but not by the large number of
cellular proteins that utilize iron cofactors. The latter forms of
iron are tightly bound in enzyme active sites and generally
should not be accessible sterically for reaction with the
hindered peroxide in FIPC-1.
We next performed competitive labeling to discern saturable
targets from high abundance proteins that might be enriched
through nonspecific labeling. In this experiment (Figure 4A),
HT-1080 cells were pretreated with DMSO or three different
concentrations (5, 15, or 50 μM) of 3 as a competitor for iron-
dependent interaction, followed 0.25 h later by the addition of
50 μM FIPC-1, and the same TMT/MS workflow described
above. Gratifyingly, the majority of enriched target proteins
showed at least partially saturable labeling (Figure 4B). Among
the most robustly competed targets were 5′-nucleotidase
domain-containing protein 2 (NT5DC2), aldose reductase
(AKR1B1), proxiredoxin-3 (PRDX3), and carbonyl reductase
(CBR1), all of which were >80% competed at 15 μM of
competitor 3 (Figure 4B). Targets competed >50% by 15 μM
3 included four members of the protein disulfide isomerase
family: protein disulfide isomerase A6 (PDIA6), protein
disulfide isomerase (P4HB), thioredoxin domain containing
5 (TXNDC5), and protein disulfide isomerase A3 (PDIA3);
glutamate dehydrogenase 1(GLUD1); transferrin receptor
protein 1 (TFRC); very long chain acyl-CoA dehydrogenase
(ACADVL); and iron−sulfur cluster assembly protein
(CIAPIN1) (Figure 4C). Certain targets like thioredoxin
(TXN) and thioredoxin reductase (TXNRD1), though
moderately enriched in the pull-down experiment, were not
competed by 3. It seems plausible that the enrichment of these
proteins may reflect their overall upregulation in response to 3
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or FIPC-1. Thus, the competition experiment in this case
serves to distinguish saturable targets of FIPC-1 from apparent
“enrichment” (false positives) arising from a cellular response
to the xenobiotic peroxide molecule itself.
TXNDC5, and PDIA3. Knockdown of individual PDIs in the
absence of ferroptosis inducer had no effect on proliferation of
HT-1080 cells. However, P4HB knockdown sensitized cells
dramatically to RSL3 and erastin, but interestingly not to
FINO2 or 3 (Figure 4E and Figure S11A). If labeling by FIPC-
1 or 3 promoted PDI dysfunction in the ER, this might be
expected to induce the unfolded protein response (UPR). To
explore this possibility, we treated HT-1080 cells with
compound 3 or a positive control inducer of the UPR,
thapsigargin, and looked for induction of the UPR effector
ATF4. Indeed, compound 3, like thapsigargin, led to ATF4
accumulation in HT-1080 cells (Figure S12A−C). Whether
UPR induction by 3 is due solely or even primarily to
interaction with PDIs cannot be discerned from these initial
experiments but is nevertheless consistent with an underex-
plored connection8 between ferroptosis and the UPR.
Since several inhibitors of P4HB have been described in the
literature, we next evaluated the effects of three such
compounds in HT-1080 cells, alone and in combination with
known ferroptosis inducers. Previously reported as PDI
inhibitors, both PACMA3143 and 35G844 (a 3-methyltoxo-
flavin) were indeed cytotoxic to HT-1080 cells, while the
compound LOC1445 was much less so (Figure 4F). To
determine whether these compounds promote ferroptotic cell
death specifically, we performed coincubations with death
suppressing compounds as before. Notably, PACMA31 toxicity
was mitigated or reversed by DFO and ferrostatin-1 but not
other death suppressors, consistent with a mechanism of
ferroptosis induction (Figure S13A−C). In contrast, 35G8-
mediated cell death could not be rescued by any of the death
suppressors, suggesting a different mechanism of action
(Figure S13A). In fact, redox cycling toxoflavins, of which
35G8 is an example, are known to catalyze the conversion of
oxygen to hydrogen peroxide46 and interfere in enzymatic
assays broadly, but especially perniciously in the case of
cysteine/GSH dependent enzymes such as PDIs. The reported
mechanism of PACMA31 action is more specific, namely,
covalent binding to the catalytic cysteine in PDIs. That this
compound, or PDI inhibition in general, can trigger ferroptosis
has not to our knowledge been previously recognized. Because
knockdown of P4HB alone failed to induce ferroptosis, the
contribution of additional PDIs or other off-targets in the
action of PACMA31 appears likely. In fact, PACMA31 was
reported47 to also inhibit PDI family members PDIA6 and
PDIA3, both of which were among the protein targets
identified with FIPC-1. While acknowledging the potential
for cross-reactivity of PACMA31 even outside the PDI family,
we note that widely used GPX4 inhibitors such as ML210 and
RSL3 also possess cysteine reactive warheads and other
structural similarities with PACMA31. Thus, the possibility
that these well-established tool compounds may have addi-
tional targets cannot be formally excluded.
Having identified several members of a FIPC-1 iron-
dependent interactome, we next considered their known
biological activities and possible roles in ferroptosis. AKR1B1
encodes an aldose reductase, a member of the family that
converts glucose to sorbital during hyperglycemia but is also
capable of reducing toxic aldehyde byproducts of lipid
peroxidation, namely 4-hydroxy-trans-2-nonenal (4-HNE)
and its glutathione adducts, to the corresponding alcohols.35,36
Similarly, the carbonyl reductase encoded by CBR1 counts
among known substrates 4-HNE as well as prostaglandins and
the quinones tocopherolquinone (vitamin E) and ubiquinone-
1 (coenzyme Q1).37,38 Thus, a protective role for AKR1B1 and
CBR1 in the later stages of ferroptosis can be readily
envisioned. Of note is that AKR1C1, a close family member
to AKR1B1, was found previously to be upregulated in erastin-
resistant cells.8 The mitochondria localized PRDX3 is a
peroxidase that catalyzes the reduction of hydrogen peroxide,
limiting availability of this species for Fenton reaction and
hydroxy radical generation.39 Finally, four members of the
protein disulfide isomerase (PDI) family were identified in the
FIPC-1 iron-dependent interactome. The PDIs are multifunc-
tional endoplasmic reticulum (ER) enzymes that mediate
disulfide formation and protein folding.40
We performed knockdown of individual target genes in HT-
1080 cells using siRNA pools targeting the specific genes.
Despite achieving over 95% knockdown of mRNA levels, we
observed only moderate impairment of cell growth with
knockdown of CBR1, AKR1B1, or PRDX3 (Figure S9A,B).
We also evaluated the effects of AKR1B1 inhibitors developed
for other clinic indication but found that none could alone
trigger ferroptotic cell death in HT-1080 cells (Figure S9C).
Given that 4-HNE should be elevated during GPX4 inhibition,
we looked for synergy between GPX4 inhibition and AKR1B1
or CBR1 knockdown (Figure S10). We found that knockdown
of CBR1, AKR1B1, or PRDX3 did not additionally sensitize
HT-1080 cells to RSL3 or indeed to any of the canonical
ferroptosis inducers studied, including 3 (Figure S10).
Paradoxically, knockdown of CBR1 or AKR1B1 protected
HT-1080 cells from erastin toxicity, an observation that might
be explained by a compensatory antioxidant response to the
KD of these genes, a possibility that merits further study.
The most robustly (∼19-fold) competed protein in the
FIPC-1 screen was the protein NT5DC2. Annotated as a
mitochondrial localized member of the haloacid dehalogenase
(HAD) superfamily,41 the functional activity and physiological
relevance of this protein remain uncharacterized. Intriguingly
then, siRNA knockdown of NT5DC2 significantly sensitized
HT-1080 cells to both RSL3 and erastin (Figure 4D and
Figure S10). In what is apparently the only previous report on
this protein, NT5DC2 was proposed to associate with and
stabilize Fyn (a proto-oncogene protein tyrosine kinase) to
promote tumor progression in glioma stem-like cells.42
Because the HAD superfamily primarily comprises phospha-
tases, further study of NT5DC2 function, its interaction with
Fyn kinase, and its larger role in the ferroptosis pathway is
warranted.
To further explore the potential utility of PACMA31 as a
ferroptosis inducer, we next asked whether the compound
exhibited synergy with canonical ferroptosis inducers erastin
and RSL3. Indeed, we found that noncytotoxic concentrations
of PACMA31 and erastin, when combined, produced a
profound killing effect in HT-1080 cells (Figure 4G). By
comparison, the combined effect of PACMA31 and RSL3 was
only additive in nature (Figure S13D). An inhibitor of cystine
import, erastin rapidly depletes the cell of an essential cofactor
(GSH) required for PDI function.48 In this way, the dramatic
synergy of system Xc− and PDI inhibition revealed herein can
Next we evaluated the effect of siRNA knockdown of four
members of the protein disulfide isomerase family identified in
the FIPC-1 interactome screen, namely PDIA6, P4HB,
F
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Notes
be understood and also suggests a potential therapeutic
combination to target ferroptosis-sensitive cells selectively.
The authors declare the following competing financial
interest(s): S.J.D. is on the scientific advisory board of Ferro
Therapeutics Inc., has consulted for Toray Industries and
AbbVie Inc., and is an inventor on patents related to
ferroptosis. A.R.R. is an advisor and co-founder of Theras,
Inc., Elgia Therapeutics, and Tatara Therapeutics.
CONCLUSIONS
■
Here we describe FIPC-1, a new tool for chemoproteomic
studies of the cellular ferrous iron-dependent interactome.
Related structurally to ferroptosis inducing peroxides with
predicted pleiotropic action, we hypothesized that FIPC-1
could identify new modulators of ferroptosis, including
ancillary players that might be missed in single-gene screening
approaches. Consistent with this, knockdown of individual
FIPC-1 target genes did not sensitize HT-1080 cells to 3 yet
did synergize with known ferroptosis inducers erastin and
RSL3 (Figure S10). Iron-dependent and competitive protein
labeling by FIPC-1 was demonstrated in a quantitative
chemoproteomic workflow that identified several saturable
protein targets, including P4HB and NT5DC2. A previously
described inhibitor of P4HB was shown to directly induce
ferroptosis and to synergize with system Xc− inhibition,
suggesting a potential therapeutic approach to selectively target
mutant-RAS driven cancers. Finally, an important role in
ferroptosis was uncovered for the uncharacterized HAD family
member NT5DC2, suggesting an unexpected new avenue in
the cellular machinery modulating ferroptosis. More broadly,
FIPC-1 and related probes based on the same design principles
are likely to find further utility in the study of diverse cellular
processes mediated or impacted by the cytosolic ferrous iron
pool.
ACKNOWLEDGMENTS
■
S.J.D. acknowledges funding from the National Institutes of
Health Research grant 1R01GM122923. A.R.R. acknowledges
funding from National Institutes of Health grant R01AI105106
and Congressionally Directed Medical Research Program,
grants W81XWH1810763 and W81XWH1810754. Mass
spectrometry was provided by the Mass Spectrometry
Resource at UCSF (A. L. Burlingame, Director) supported
by the Dr. Miriam and Sheldon G. Adelson Medical Research
Foundation (AMRF) and the UCSF Program for Break-
through Biomedical Research (PBBR). We thank F. Mc-
Cormick and E. Collisson (UCSF) for providing GPX4 KO
A375 cells and R. Perera (UCSF) for the inducible KRAS
(G12D) AK210 cells. We thank D. Medina-Cleghorn for
experimental advice and L. Magtanong for help with imaging
experiments. We acknowledge B. Blank and P. Talukder for
synthesizing arterolane and artefenomel.
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ASSOCIATED CONTENT
* Supporting Information
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AUTHOR INFORMATION
Corresponding Author
Adam R. Renslo − Department of Pharmaceutical Chemistry,
University of California, San Francisco, San Francisco,
California 94143, United States; orcid.org/0000-0002-
1240-2846; Email: adam.renslo@ucsf.edu
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Ying-Chu Chen − Department of Pharmaceutical Chemistry,
University of California, San Francisco, San Francisco,
California 94143, United States
Juan A. Oses-Prieto − Department of Pharmaceutical
Chemistry, University of California, San Francisco, San
Francisco, California 94143, United States
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Stanford, California 94305, United States
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Alma L. Burlingame − Department of Pharmaceutical
Chemistry, University of California, San Francisco, San
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Scott J. Dixon − Department of Biology, Stanford University,
Stanford, California 94305, United States; orcid.org/0000-
0001-6230-8199
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