Diacylfuroxans Are Masked Nitrile Oxides That Inhibit GPX4 Covalently

Article abstract of DOI:10.1021/jacs.9b10769

GPX4 represents a promising yet difficult-to-drug therapeutic target for the treatment of, among others, drug-resistant cancers. Although most GPX4 inhibitors rely on a chloroacetamide moiety to modify covalently the protein's catalytic selenocysteine residue, the discovery and mechanistic elucidation of structurally diverse GPX4-inhibiting molecules have uncovered novel electrophilic warheads that bind and inhibit GPX4. Here, we report our discovery that diacylfuroxans can act as masked nitrile oxide prodrugs that inhibit GPX4 covalently with unique cellular and biochemical reactivity compared to existing classes of GPX4 inhibitors. These observations illuminate a novel molecular mechanism of action for biologically active furoxans and also expand the collection of reactive groups capable of targeting GPX4.

Full text of DOI:10.1021/jacs.9b10769

Article
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Diacylfuroxans Are Masked Nitrile Oxides That Inhibit GPX4
Covalently
John K. Eaton,† Richard A. Ruberto,† Anneke Kramm,† Vasanthi S. Viswanathan,*,†
and Stuart L. Schreiber*,†,‡
†Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, United States
‡Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
S
* Supporting Information
ABSTRACT: GPX4 represents a promising yet difficult-to-
drug therapeutic target for the treatment of, among others,
drug-resistant cancers. Although most GPX4 inhibitors rely on
a chloroacetamide moiety to modify covalently the protein’s
catalytic selenocysteine residue, the discovery and mechanistic
elucidation of structurally diverse GPX4-inhibiting molecules
have uncovered novel electrophilic warheads that bind and inhibit GPX4. Here, we report our discovery that diacylfuroxans can
act as masked nitrile oxide prodrugs that inhibit GPX4 covalently with unique cellular and biochemical reactivity compared to
existing classes of GPX4 inhibitors. These observations illuminate a novel molecular mechanism of action for biologically active
furoxans and also expand the collection of reactive groups capable of targeting GPX4.
may be useful not only for targeting GPX4 but more broadly
for the development of covalent chemical biology tools for
other proteins.
INTRODUCTION
■
Glutathione peroxidase 4 (GPX4) plays a critical role in the
dissipation of cellular lipid oxidative stress.1,2 Among
antioxidant proteins, GPX4 has the unique ability to reduce
complex lipid hydroperoxides,3,4 the accumulation of which
leads to ferroptotic cell death.1,5 The direct inhibition of GPX4
enzymatic function is an effective means to induce ferroptosis
in certain cellular contexts,1,6,7 most notably to kill cancer cells
that are in a pan-therapy-resistant state.6−8
In our continuing pursuit to catalog chemotypes that are
able to bind and inhibit GPX4, we investigated a small subset
of compounds with NCI-60 cell line sensitivity profiles that
correlate with known ferroptosis inducers.18 One of these
compounds is NSC144988 (1), which contains an unusual
furoxan heterocycle (Figure 2A). Here, we report our
discovery that 1 and related diacylfuroxans represent a novel
class of masked nitrile oxide electrophiles that covalently target
GPX4 and other (seleno)cysteine-containing proteins.
GPX4 is challenging to inhibit with small molecules because
it lacks a well-defined active site or known allosteric regulatory
sites.9,10 To date, the only cell-active GPX4 inhibitors that have
been reported rely on reactive warheads that covalently engage
the catalytic selenocysteine (Sec, U) residue of GPX4.1,10,11
Notably, nearly all validated GPX4 inhibitors, such as RSL3,
ML162, and DPI17 (Figure 1A), rely on a chloroacetamide
warhead or related chloromethyltriazine group.1,12 Despite the
high nucleophilicity of the selenocysteine selenol,13,14 it has
not been possible to replace the chloroacetamide functionality
of GPX4 inhibitors with less reactive alternatives.1,10,15
Furthermore, reported cellular1,16 and biochemical9 screens
have not identified other reactive groups capable of inhibiting
GPX4. The limitations of chloroacetamide-containing inhib-
itors, including low target selectivity and poor pharmacokinetic
properties,10,17 have motivated us to identify GPX4-targeting
small molecules with alternative electrophilic chemotypes.
We have recently reported that an unusual class of masked
nitrile oxide electrophiles, exemplified by ML210, JKE-1674,
and JKE-1716 (Figure 1A,B) can target GPX4 selectively in
cells.10 On the basis of these findings, we hypothesized that
GPX4 represents a promising model system for identifying
additional novel masked electrophilic warheads that rely on
unexpected cellular chemical transformations. Such warheads
RESULTS
■
Diacylfuroxans Are Inducers of Ferroptotic Cell
Death. To determine if diacylfuroxans, exemplified by 1 and
related analogs 2 and 3 (Figure 2A), induce ferroptosis, we
assessed their effects on the viability of cells in the presence
and absence of ferrostatin-1 (fer-1), a lipophilic radical-
trapping antioxidant that inhibits ferroptosis.5,19,20 The cell-
killing activity of 1−3 was suppressed by cotreatment with fer-
1 (Figures 2B,C and S1A) and other ferroptosis inhibitors5,21
(Figure S1B). Treatment of cells with 1−3 led to the
accumulation of cellular lipid hydroperoxides that could be
prevented by fer-1 cotreatment (Figures 2D and S1C),
indicating that diacylfuroxans are indeed capable of inducing
ferroptotic cell death.
Ferroptotic cell death can be induced not only by direct
inhibition of GPX4 but also through the inhibition of upstream
targets in the glutathione biosynthesis pathway.22,23 To rule
Received: October 12, 2019
© XXXX American Chemical Society
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Figure 1. Summary of small-molecule GPX4 inhibitors. (A) Structures of selected small-molecule GPX4 inhibitors. Chloroacetamide and
chloromethyltriazine groups of RSL3, ML162, and DPI17 are shown in red. Masked nitrile oxide groups of ML210, JKE-1674, and JKE-1716 are
shown in blue. (B) Proposed pathway for the unmasking of nitrile oxide electrophiles in cells.
Figure 2. Diacylfuroxans induce ferroptosis. (A) Structures of diacylfuroxans 1 (NSC144988), 2, and 3. The furoxan group (1,2,5-oxadiazole 2-
oxide) is highlighted in blue. (B) Cotreatment with fer-1 (1.5 μM) rescues the cell-killing effects of 1, 2, and 3 in LOX-IMVI cells. Data are plotted
as the mean s.e.m., with n = 4 technical replicates. (C) Summary of diacylfuroxan activity and selectivity in cancer cell lines. aLOX-IMVI, bKP4,
and cPANC02. See also Figure S1A. (D) Treating cells with GPX4 inhibitors (RSL3 and ML210; 10 μM, 1 h) or diacylfuroxans 1 and 2 (20 μM, 1
h) leads to the accumulation of lipid hydroperoxides as assessed by C11-BODIPY 581/591 fluorescence in LOX-IMVI cells. The C11-BODIPY dye
emission shifts from orange to green upon oxidation. Fer-1 cotreatment (1.5 μM) prevents lipid hydroperoxide accumulation. Scale bars, 50 μm.
See also Figure S1C.
out that diacylfuroxans may be inhibiting glutathione biosyn-
thesis, we assessed the effects of these compounds on cellular
glutathione. We found that treating cells with 1−3 did not
perturb glutathione levels to the extent expected for
glutathione biosynthesis inhibitors such as erastin1,24 (Figure
S1D). We also ruled out the prospect that nitric oxide (NO)
donation, a common mechanism of action of furoxan-
containing compounds,25−27 underlies the ferroptosis-inducing
activity of 1−3 (Figure S2). These observations left open the
possibility that diacylfuroxans induce ferroptosis by targeting
GPX4 directly.
B
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Figure 3. Diacylfuroxans target GPX4 covalently. (A) Structures of diacylfuroxan affinity probes 4 and 5 and cellular activity in LOX-IMVI cells.
See also Figure S3A,B. (B) GPX4 pulldown by diacylfuroxan affinity probes 4 and 5 (20 μM, 1 h) after cell treatment. (C) GPX4 pulldown by 5
(20 μM, 30 min) can be blocked by pretreatment with GPX4 inhibitors (10 μM, 30 min). (D) Structure of the RSL3-yne affinity probe with the
alkyne group shown in blue. (E) Pulldown of GPX4 by RSL3-yne (10 μM, 30 min) can be blocked by the pretreatment of cells with 2 (20 μM, 30
min). See also Figure S3C.
Figure 4. Diacylfuroxans bind GPX4 via a nitrile oxide intermediate. (A) Diacylfuroxan 2 reacts with thiols to yield adducts containing a
thiohydroximate structure. (B) The hydrolysis of 2 yields nitrile oxide 8 with the loss of 4-methylbenzoic acid. (C) Cotreatment with fer-1 (1.5
μM) rescues the cell-killing effects of 8 in LOX-IMVI melanoma cells. Data are plotted as the mean s.e.m., with n = 4 technical replicates. (D and
E) Incubation of GPX4U46C allCys(−) (5 μM) with diacylfuroxan 2 (50 μM, 1 h) produces a +204 Da adduct based on intact protein mass
spectrometry. (F) Incubation of GPX4U46C allCys(−) (5 μM) with nitrile oxide 8 (50 μM, 1 h) produces the same +204 Da adduct as 2. (G)
Structure of proposed thiohydroximate adduct derived from 2 and 8.
Diacylfuroxans Bind GPX4 Covalently. To determine if
diacylfuroxans induce ferroptosis by binding and inhibiting
GPX4 directly, we developed two affinity probe derivatives of
1−3 containing azide (4) or alkyne (5) groups (Figure 3A).
These analogs have cellular potency similar to that of 1−3 and
retain the ability to induce ferroptotic cell death (Figures 3A
and S3A,B). Both 4 and 5, like established GPX4 inhibitor
affinity probes,10 enable the direct pulldown of GPX4 from
cells after conjugation to the appropriate alkyne- or azide-
biotin reagent via copper(I)-catalyzed azide−alkyne cyclo-
addition (CuAAC) chemistry,28 affinity enrichment with solid-
supported streptavidin, and visualization by SDS-PAGE and
western blotting (Figure 3B). The ability of these probes to
pull down GPX4 in this assay under denaturing conditions and
to induce a GPX4 gel shift, in a manner similar to that of
established irreversible GPX4 inhibitors,10 is consistent with
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Figure 5. Diacylfuroxan structure−activity relationship (SAR) studies. (A) Analogs of diacylfuroxans 2 and 3. Modifications to the acyl groups are
highlighted in blue. (B) Summary of diacylfuroxan and 4-nitroisoxazole activity and selectivity in LOX-IMVI cells. See also Table S1 and Figures S7
and S8. (C) Proposed mechanism for diacylfuroxan hydrolysis to generate a nitrile oxide and carboxylic acid.
diacylfuroxans acting as covalent GPX4 inhibitors. Compound
5 is unable to engage GPX4 in cells pretreated with
selenocysteine-binding GPX4 inhibitors10,12,29 (Figure 3C),
suggesting that diacylfuroxans also interact covalently with the
selenocysteine residue.
To characterize the interaction between GPX4 and
diacylfuroxans further, we performed competitive GPX4
pulldown experiments with RSL3-yne (Figure 3D),10 an
alkyne probe derived from selenocysteine-binding GPX4
inhibitor RSL3.12,29 We found that the pretreatment of cells
with diacylfuroxans blocks GPX4 pulldown by RSL3-yne,
which is otherwise able to enrich GPX4 in the absence of
competitors (Figures 3E and S3C). These observations
confirm the mutually exclusive nature of GPX4 binding by
diacylfuroxans and other GPX4 inhibitors and support the fact
that the catalytic selenocysteine residue is the binding site for
diacylfuroxans.
Diacylfuroxans Bind GPX4 via a Nitrile Oxide Electro-
phile. To investigate the mechanism by which diacylfuroxans
covalently bind GPX4, we initially assessed the reactivity of 2
toward nucleophiles. We found that thiols react directly with 2
to form covalent adducts with the concomitant loss of a single
4-toluoyl group, exemplified by thiohydroximate adducts 6 and
7 (Figure 4A). This transformation is strongly suggestive that a
nitrile oxide intermediate is the active species derived from 2,
and the structure of these adducts is similar to those formed by
the active nitrile oxide electrophile derived from ML210 and
related nitrile oxide precursors (e.g., JKE-1674).10
We found that the putative nitrile oxide species derived from
2, compound 8, could be isolated along with byproduct p-
toluic acid following the treatment of 2 with aqueous base
(Figure 4B). This hydrolysis of 2 occurs in aqueous solutions
including PBS buffer (pH 7.4) (Figure S4A) and can be
controlled by adjusting the solution pH (Figure S4B). The rate
of diacylfuroxan hydrolysis increases with increasing solution
basicity but can be suppressed completely in acidic buffers
(Figure S4B). We confirmed that other ferroptosis-inducing
diacylfuroxans are similarly hydrolyzed (Figure S4C). The
isolation of compound 8 is noteworthy because while this type
of conversion of diacylfuroxans to nitrile oxides has been
inferred from their previously reported reactivity with
dipolarophiles30−32 or nucleophiles,33 the nitrile oxide
intermediates were not observed directly in these cases.
Several observations support that diacylfuroxan 2 reacts via
nitrile oxide 8 to inhibit GPX4 and induce ferroptotic cell
death. First, 8 exhibits the ability to induce ferroptosis in LOX-
IMVI cells with a potency and degree of fer-1 rescue similar to
those of 2 (Figure 4C). Second, 8 forms thiol adducts which
are identical to those formed by 2 (Figure S5A−C). These
adducts are not able to induce ferroptosis in cells (Figures 4D
and S5D,E) and therefore do not explain the cellular effects of
2. Finally, 2, nitrile oxide 8, and other diacylfuroxans react with
purified GPX4U46C allCys(−) protein (Figure S6A; see
Materials and Methods) to produce adducts with masses
consistent with thiohydroximate structures (Figures 4E,F and
S6). These results strongly suggest that diacylfuroxan-derived
nitrile oxides bind the catalytic (seleno)cysteine 46 residue of
GPX4 because GPX4U46C allCys(−) contains only a single
cysteine residue at this position. The observation that GPX4
binding by diacylfuroxans and established (seleno)cysteine-
binding inhibitors is mutually exclusive (Figure 3C,E) further
supports that the active-site (seleno)cysteine residue interacts
with diacylfuroxan-derived nitrile oxides covalently.
Structure−Activity Relationship (SAR) Studies of
Diacylfuroxans. Although compounds containing furoxan
heterocycles are known to be reactive in biological
contexts,25,26 the ability of diacylfuroxans to act as masked
nitrile oxides has not been described previously. Structure−
activity relationship (SAR) studies of diacylfuroxans (Figure
5A,B and Table S1) revealed the critical role of the acyl groups
during the transformation of diacylfuroxans into nitrile oxide
electrophiles. We found that a variety of diacyl-substituted
furoxans (15, 17−20) exhibit the ability to induce ferroptosis
(Table S1) by targeting GPX4 (Figure S6G). In contrast,
furoxan analogs lacking acyl groups (9−12) (Figure 5A) show
no ability to target GPX4 as determined by cellular lipid
peroxidation and ferroptotic cell death assays (Figures 5B and
S7). One exception to this trend is structurally similar
disulfonylfuroxan 13 (Figure 5A), which we found binds
GPX4 covalently (Figure S8A,B) and rapidly depletes cellular
glutathione levels (Figure S8C). Despite these observations, 13
reacts in a manner chemically distinct from that of
diacylfuroxans and does not involve a nitrile oxide intermediate
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Figure 6. Chemoproteomic characterization of diacylfuroxans. (A) Assessment of the proteome-wide reactivity of 4 in LOX-IMVI cells by ABPP
(1 h treatment). See Figure S10B for an assessment of probe 5. (B) Pretreatment of LOX-IMVI cells with 2 (30 min) blocks, in a concentration-
dependent manner, the labeling of proteins by 4 (5 μM, 30 min). See Figure S10D for an assessment of probe 5. (C) Comparison of cellular
reactivity profiles of GPX4-inhibitor probes (10 μM, 1 h) in LOX-IMVI cells. See also Figure S11A−C. (D) Proteins enriched specifically by
diacylfuroxan 5. See also Table S2. Listed proteins were enriched ≥5-fold vs the vehicle (DMSO) and ≥5-fold vs both RSL3-yne and ML162-yne.
(E) Validation of selected proteins targets of 5 by western blotting. See also Figure S12.
(Figure S8D).34,35 The ability of 13 to promote lipid
peroxidation and kill cells cannot be suppressed efficiently by
fer-1 cotreatment (Figure S8E,F), consistent with its extensive
and nonselective proteome-wide reactivity (Figure S8G).
These findings highlight the diverse reactivity that is possible
for furoxans and reinforce the unique ability of diacylfuroxans
among this class of heterocycles to act as masked nitrile oxides
(Figure 5C).
Another finding from our SAR studies is that 4-nitro-
isoxazoles 21−23 (Figure S9A), which are isomeric with
diacylfuroxans 1−3, respectively, are unable to target GPX4 or
induce ferroptosis as assessed by competitive binding experi-
ments (Figure S3C), cell viability assessments with fer-1
cotreatment (Figure S9B), and lipid peroxidation measure-
ments (Figure S9C). These 4-nitroisoxazoles arise as by-
products from common procedures used to synthesize
diacylfuroxans (Figure S9D),36,37 and their inability to induce
ferroptosis is notable given the structural similarity with
nitroisoxazole-containing ML210. The inactivity of 21−23
may reflect the poorly understood SAR trends underlying the
cellular transformation of 4-nitroisoxazoles into GPX4-
targeting nitrile oxides. These observations also underscore
the different chemical mechanisms involved in the unmasking
of 4-nitroisoxazole and diacylfuroxan heterocycles to reveal
nitrile oxide electrophiles in cells.
Diacylfuroxans Exhibit Distinct Proteome-Wide Re-
activity in Cells Compared to Other GPX4 Inhibitors.
Our findings reveal that diacylfuroxans are readily converted
into electrophilic nitrile oxides with high reactivity toward thiol
and selenol nucleophiles. Because of this reactivity, we
expected that diacylfuroxans would interact with many proteins
in cells beyond GPX4. To explore the cellular proteome-wide
reactivity of this compound class, we used diacylfuroxan
affinity probes 4 and 5 to perform activity-based protein
profiling (ABPP)38 experiments. These experiments involve
the treatment of intact cells or cell lysates with affinity probes,
subsequent CuAAC conjugation with azide- or alkyne-bearing
fluorescent dyes, and the visualization of probe-labeled
proteins by fluorescence imaging of SDS-PAGE gels. We
observed that both 4 (Figures 6A and S10A) and 5 (Figure
S10B) label cellular proteins in a dose-dependent manner and
that their overall patterns of labeling are similar. The labeling
of several prominent proteins (e.g., at ∼42 kDa) could be
blocked by the pretreatment of cells with 2, suggesting specific
binding interactions,39 while other proteins were insensitive to
pretreatment with 2, indicating nonspecific protein labeling
(Figures 6B and S7C,D).
Diacylfuroxan 5 exhibits a distinct pattern of proteome
reactivity in cells (Figures 6C and S11B,C) compared to
chloroacetamide (RSL3-yne, ML162-yne) and ML210-family
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probes (ML210-yne, JKE-1674-yne) (Figure S11A). Com-
petitive labeling experiments further corroborate that the major
protein targets of 5 are not shared with other GPX4 inhibitors
(Figure S11D). To identify the protein targets of 5, RSL3-yne,
and ML162-yne in cells, we performed mass spectrometry
(MS)-based chemoproteomics experiments (Figures 6D and
S12A and Table S2). Although we were unable to detect GPX4
in these mass spectrometry- and gel-based ABPP studies, likely
because of the relatively low expression of this protein, we
confirmed the enrichment of GPX4 in the same samples by
western blotting (Figures 6E and S12B).
Notably, nearly all proteins enriched by 5 (Figure 6D, Table
S2) are known to contain ligandable (seleno)cysteine
residues,29,40,41 and we validated a subset of these proteins
by western blotting (Figures 6E and S12B,C). The top protein
enriched specifically by 5, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; ∼42 kDa band in Figure 6A−C),
likely reacts via its catalytic cysteine residue, which has been
previously identified as ligandable40 and is known to bind
the covalent inhibitor koningic acid.42 Other highly enriched
proteins identified as unique targets of 5 are members of the
protein disulfide-isomerase (PDI) family, including PDIA4,
PDIA3, P4HB (PDI), and PDIA6 (Figure 6D and Table S2),
which contain multiple ligandable cysteine residues.40
Although PDI is a known target of both RSL31 and structurally
similar compounds,43 our findings indicate that 4 and 5
interact with PDI to a greater extent compared to other GPX4
inhibitors. The prevalence of (seleno)cysteine-containing
proteins enriched by 5 suggests that proteins are bound
covalently by a 5-derived nitrile oxide to form seleno- or thio-
hydroximate adducts in a manner consistent with our
diacylfuroxan reactivity studies with GPX4. It is possible that
some proteome-wide reactivity is due to different chemical
reactions of diacylfuroxans (e.g., protein benzoylation) or the
labeling of non(seleno)cysteine residues, although we found
no examples of such reactivity.
JKE-1674-yne, does not interact readily with GPX4 in the cell
lysate under the assay conditions (Figure S13B) despite the
greater cellular potency of RSL3-yne (>100-fold) compared to
that of 5 (Figure S13C). We found previously that RSL3
exhibits biochemical activity that is weaker than expected given
its cellular potency, including reactivity toward GSH and the
ability to bind wild-type GPX4 protein.10 Consistent with our
findings, a recent study reported that RSL3 does not efficiently
bind purified GPX4 without the assistance of an adaptor
protein.46 These observations highlight the limitations of
established small-molecule GPX4 inhibitors as biochemical
probes and suggest that 5 and other diacylfuroxans may have
unique advantages due to their greater ability to react with
GPX4 outside of the cellular context.
DISCUSSION
■
We have discovered that diacylfuroxans can act as masked
nitrile oxide electrophiles to target GPX4 covalently and
induce ferroptotic cell death. Our discovery illuminates
diacylfuroxans as a novel class of masked electrophilic
warheads with unique reactivity properties, both biochemically
and in intact cells, making them useful tool compounds for
studying GPX4. The ability to study the effects that small-
molecule inhibitors have on GPX4 has been hindered by both
the difficulty of obtaining selenocysteine-containing wild-type
GPX4 protein3,10,47 and the lack of structurally diverse tool
compounds with activity in biochemical assays. Because
diacylfuroxans interact more readily with GPX4 outside of
the cellular context compared to established inhibitors (e.g.,
RSL3 and ML210), they may enable the determination of
cocrystal structures with GPX4 to better understand the
structural basis of GPX4-inhibitor binding. Such information
may help to elucidate the discrepancy we have noted between
the potency of GPX4 inhibitors in cellular versus biochemical
contexts and may provide useful information for the rational
design of improved GPX4 inhibitors.
Despite the unique cellular reactivity profile of diacylfurox-
ans, there are common targets with chloroacetamide-based
probes that further reinforce the expected (seleno)cysteine
reactivity of diacylfuroxans. For example, probes 4, 5, RSL3-
yne, and ML162-yne enrich selenoprotein thioredoxin
reductase 1 (TXNRD1) (Figure S12B,C). A recent report
identified the TXNRD1 selenocysteine residue as the binding
site of RSL3,29 an observation that indicates that 4, 5, and
ML162-yne may also bind at this site. Similarly, diacylfuroxan
probes enrich glutathione S-transferase omega 1 (GSTO1)
(Figure S12C), which binds ML162 and other chloroaceta-
mides via its active-site cysteine residue.44,45 This overlap
between targets of diacylfuroxans and chloroacetamide-based
GPX4 inhibitors further supports that the cellular reactivity of
diacylfuroxans is due to the binding of the (seleno)cysteine
residue via their active nitrile oxide forms.
Diacylfuroxans Exhibit Distinct Proteome-Wide Re-
activity in Cell Lysates Compared to Other GPX4
Inhibitors. ABPP experiments in cell lysates highlight another
distinct reactivity of diacylfuroxans compared to other classes
of GPX4 inhibitors. Although diacylfuroxan 5 exhibits
comparable proteome labeling in both cells and cell lysates,
chloroacetamide- and ML210-derived probes display reactivity
in cell lysates that is diminished compared to their reactivity in
intact cells (Figure S13A). The discrepancy between
compound reactivity in intact cells versus lysates is especially
notable for RSL3-yne which, like prodrugs ML210-yne and
On the basis of our investigation of diacylfuroxans as well as
our previous studies of ML210,10 it appears that the masking
elements of prodrug nitrile oxides do not necessarily contribute
directly to their selectivity in cells. Rather, these features confer
nitrile oxides, which are inherently unstable due to multiple
decomposition pathways,10,48,49 with the stability necessary for
routine use in biochemical and cellular experiments. This is
similar conceptually to the masked covalent inhibitor
omeprazole and related compounds, which are prodrug
proton-pump inhibitors requiring conversion to their active
sulfenamide and sulfenic acid forms under acidic condi-
tions.50−52 Like nitrile oxides, the decomposition of these
sulfenamides and sulfenic acids yields a mixture of inactive
products,50,53 and it would not be possible to deliver these
compounds if not for their more stable masked prodrug forms.
A variety of structurally diverse masked electrophiles have
been reported previously,54−59 many of which rely on
nonenzymatic activation, such as diacylfuroxans and omepra-
zole. However, the enzymatic formation of nitrile oxides has
been reported in the context of drug metabolism60 and natural
product biosynthesis,61,62 suggesting that there may be
additional strategies for developing prodrugs that act via nitrile
oxides in cells. More broadly, diverse methods for masking the
reactivity of nitrile oxides may enable the development of
improved covalent inhibitors of GPX4 and other proteins.
Taken together, our observations of diacylfuroxans reinforce
our recent discovery that masked nitrile oxides may be well
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suited for targeting GPX4 and broaden the scope of specific
chemotypes available for delivering highly reactive nitrile oxide
electrophiles to cells. The unique in situ reactivity and
structural diversity of masked nitrile oxides including 4-
nitroisoxazoles, α-nitroketoximes, nitrolic acids, and now
diacylfuroxans, warrant additional study and exploration as
electrophilic probes of cellular circuitry.
CONCLUSIONS
■
Diacylfuroxans represent a new chemotype capable of targeting
GPX4 in cells to induce ferroptosis. Mechanistically,
diacylfuroxans act as masked nitrile oxides that are activated
under aqueous conditions. Compared to established small-
molecule GPX4 inhibitors, diacylfuroxans possess unique
reactivity properties in biochemical and cellular contexts. We
anticipate that diacylfuroxans and other masked nitrile oxides
will serve as promising chemical tools for the study of GPX4
and other protein targets.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b10769.
■
S
Materials and methods, figures and tables, and NMR
spectra (PDF)
Label-free quantification of proteins enriched from
LOX-IMVI cells and summary of proteins enriched
selectively by diacylfuroxan 5 (XLSX)
(9) Sakamoto, K.; Sogabe, S.; Kamada, Y.; Matsumoto, S.; Kadotani,
A.; Sakamoto, J.; Tani, A. Discovery of GPX4 Inhibitory Peptides
from Random Peptide T7 Phage Display and Subsequent Structural
Analysis. Biochem. Biophys. Res. Commun. 2017, 482 (2), 195−201.
(10) Eaton, J. K.; Furst, L.; Ruberto, R. A.; Moosmayer, D.; Hillig, R.
C.; Hilpmann, A.; Zimmermann, K.; Ryan, M. J.; Niehues, M.;
Kramm, A. et al. Targeting a Therapy-Resistant Cancer Cell State
Using Masked Electrophiles as GPX4 Inhibitors. bioRxiv 2018,
DOI: 10.1101/376764.
AUTHOR INFORMATION
Corresponding Authors
*vasanthi@broadinstitute.org
*stuart_schreiber@harvard.edu
ORCID
John K. Eaton: 0000-0003-4633-5546
Stuart L. Schreiber: 0000-0003-1922-7558
Notes
The authors declare the following competing financial
interest(s): S.L.S. is a member of the Board of Directors of
the Genomics Institute of the Novartis Research Foundation
(GNF); a shareholder and member of the Board of Directors
of Jnana Therapeutics; a shareholder of Forma Therapeutics; a
shareholder of and adviser to Decibel Therapeutics and
Eikonizo Therapeutics; an adviser to Eisai, Inc., the Ono
Pharma Foundation, and F-Prime Capital Partners; and a
Novartis Faculty Scholar.
■
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ACKNOWLEDGMENTS
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This work was supported by grants from the U.S. National
Institutes of Health (NIH R01GM038627 and
R35GM127045). We thank Bogdan Budnik and Renee
Robinson of the Harvard University Mass Spectrometry and
Proteomics Resource Laboratory for proteomics support,
Nadine Elowe and Jamie Moroco for technical assistance
with intact protein mass spectrometry experiments, and Laura
Furst for helpful discussions.
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