Diverse compounds from pleuromutilin lead to a thioredoxin inhibitor and inducer of ferroptosis

Article abstract of DOI:10.1038/s41557-019-0261-6

The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.

Full text of DOI:10.1038/s41557-019-0261-6

Articles
https://doi.org/10.1038/s41557-019-0261-6
Diverse compounds from pleuromutilin lead to a
thioredoxin inhibitor and inducer of ferroptosis
1
1
1
1
2
Evijola Llabani , Robert W. Hicklin , Hyang Yeon Lee , Stephen E. Motika , Lisa A. Crawford ,
2
1
Eranthie Weerapana and Paul J. Hergenrotherꢀ ꢀ *
The chemical diversification of natural products provides a robust and general method for the creation of stereochemically
rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in
most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natu-
ral product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort
resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and
highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel
compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identifica-
tion efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxi-
dant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a
useful tool to study the utility of pro-ferroptotic agents for treatment of cancer.
tructurally complex small molecules play an important role in immunostimulatory activity in a murine cancer model and thus will
probing biological systems and combating disease. Such com- be an important tool for further investigating the potential of fer-
S
pounds often contain dense polycyclic ring systems, multiple roptosis-inducing agents to act in concert with the immune system
stereogenic centres and spatially defined arrangements of func- as an anticancer strategy.
tional groups. The complexity and three-dimensionality of these
molecules allow for specific interactions with biological macromol- Results and discussion
ecules and selective modulation of cellular pathways; such com- Diversifying pleuromutilin. The diterpene natural product P is
pounds are complementary to those in large commercial screening found in several species of fungi and is a potent inhibitor of the
18
collections that tend to have fewer stereogenic centres and more bacterial 50S ribosome . P is composed of five-, six- and eight-
2
1,2
sp -hybridized carbons .
membered rings and contains eight contiguous stereogenic centres.
Several strategies for the rapid and efficient synthesis of value- Several semisynthetic derivatives of P are used to treat Gram-
added complex compounds have been developed, including those positive pathogens in humans (retapamulin) and in veterinary
18
that construct complex scaffolds from diverse collections of simple medicine (tiamulin, valnemulin) , and epi-mutilin derivatives have
3,4
building blocks and those that begin with complexity and build in recently been developed as antibiotics with activity against some
5
19–22
diversity . For this latter approach, natural products offer a rich and Gram-negative bacteria . Investigation of the antibacterial activ-
varied source of starting materials, and collections of compounds ity of P and its derivatives has inspired several total synthesis effo
2
0,21,23–25
have been assembled using the complexity-to-diversity (CtD) strat- rts
that, combined with previous work on structure elucida-
6
6
26,27
18,28,29
egy from the natural products adrenosterone , gibberellic acid , tion
and structure–activity relationship studies
, provide a
6,7
8
9
10
11
quinine , abietic acid , sinomenine , lycorine , yohimbine , hae- wealth of synthetic information about the chemical reactivity of the
12
manthamine , nitrogenous steroids of dutasteride and abiraterone pleuromutilin ring system. Structural transformations of the P ring
13
14
acetate , ilimaquinone and others. The resulting collections have system identified through these efforts afford several good start-
been used to discover small molecules with anticancer and antimi- ing points for novel diversification reactions. With the objective of
11,15
16
crobial activities , autophagy inhibitors and to identify predic- harnessing the advantages of P as a starting material to construct a
17
tive guidelines for broad-spectrum antibiotic discovery .
small set of highly complex and structurally diverse compounds, we
The CtD strategy is applied here to the natural product pleu- set out to alter the ring systems of pleuromutilin through a series of
romutilin (P, Fig. 1) with an emphasis on transforming the highly ring contraction, expansion, cleavage and fusion reactions (Fig. 1).
dense ring system of P into compounds with novel and complex
Treatment of pleuromutilin with phosphorus pentachloride
ring architectures in short synthetic sequences. The resulting com- results in activation of the secondary alcohol, carbocation rear-
2
6
pounds were then evaluated for their ability to induce rapid death rangement and ring contraction to form known diene P1 (Fig. 1a)
of cancer cells, with an eye toward the discovery of compounds with as a single diastereomer; its absolute configuration was confirmed
unusual modes of action. We now report the identification of fer- by X-ray crystallography (Supplementary Table 1). P1 is an out-
roptocide, a novel compound that induces rapid ferroptotic death standing starting point for the construction of novel compounds
of cancer cells and inhibits thioredoxin; its mechanism of ferrop- with unusual oxidation patterns and ring systems. For example,
totic induction makes ferroptocide distinct from and complemen- silylation of P1 results in formation of the P2 kinetic silyl enol
tary to existing ferroptosis inducers. Additionally, ferroptocide has ether. Subsequent Rubottom oxidation induces an alcohol-directed
1
2
Department of Chemistry, Roger Adams Laboratory, University of Illinois, Urbana, IL, USA. Department of Chemistry, Boston College, Chestnut Hill,
MA, USA. *e-mail: hergenro@illinois.edu
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Articles
NaTuRe CHemISTRy
mCPBA
AcOH
TBSOTf
Et3N
O
O
O
O
O
PCl5
8%
Pyridine
Et3N·3HF
Pb(OAc)4
HO
a
H
H
O
O
O
O
O
5
CH2Cl2
CH2Cl2
40%
THF
76%
Benzene
O
Cl
Cl
Cl
Cl
Cl
9
9%
MeOH
O
TBSO
O
OH
OH
O
TBSO
O
43%
O
P1
P2
P3
P4
P5
Ring expansion
Ring contraction
(
i) CH(OMe)3
H2SO4
O
O
O
O
mCPBA
MeOH
PCl5
NaHCO3
MeMgBr
b
c
(
ii) NaOH, 73%
2 steps)
HO
O
Pentane
Benzene
H
O
CH2Cl2
67%
H
O
THF
60%
H
O
(
H
H
O
H
HO
2
3%
P6
P7
P8
P9
Ring expansion
OH
O
O
O
O
O
O
NH2OH·HCl
NaOAc
H
(
i) PCC, 84%
(ii) KOH
O
Cyanuric chloride
O
OH
O
O
N
OH
EtOH
59%
DMF
52%
(
iii) PCC, CH2Cl2
6% (2 steps)
NH
O
4
Pleuromutilin (P)
P10
P11
P12
Ring cleavage
Ring fusion
Ring expansion
(
i) NaOH, 92%
ii) Ac2O
DMAP, 68%
OAc
AgNO3
OAc
O
t
H
N
H
(
Bu3tpy
O2
t
N
NH2
H
O
PhI(OAc)2
O
BuOK
O
H
H
d
e
t
O
(
iii) ClSO2NCO
CH2Cl2
O
CH3CN
76%
O
BuOH
55%
O
7
6%
O
O
O
P13
P14
Ring fusion
P15
Ring expansion
OH
H
4
steps
PCl5
2
H
H
CH Cl2
5
1%
O
O
OH
P17
Ring rearrangement
P16
Ring fusion
Fig. 1 | Compounds synthesized via ring system distortion of pleuromutilin using the CtD strategy. a, Synthetic route to P5 from P using a ring
contraction of the eight-membered ring followed by a Rubottom oxidation and oxidative cleavage. b, Synthetic route to P9 from P using ring expansion,
diastereoselective epoxidation and elimination. c, Synthetic route to lactam P12 from P using a retro-Michael ring cleavage and oxidative rearrangement
followed by a Beckmann ring expansion. d, Synthetic route to P15 upon ring fusion of P by C–H amidation followed by alkaline autoxidation, hydride
33
migration and lactonization. e, Synthesis of oxafenestranes from P as described previously .
epoxidation on the less hindered face of the five-membered ring, oxidation of the resulting ketal with pyridinium chlorochromate,
24
yielding epoxide P3 as a single diastereomer (Fig. 1a). Desilylation provides known lactone P10 (Fig. 1c) . Compound P10 was then
of P3 to P4 (Fig. 1a) provides an α-hydroxy ketone that, upon expo- used to construct novel lactam P12 through ring expansion of the
sure to lead tetraacetate, produces a novel rearrangement yielding cyclopentanone ring of P10 induced by oxime formation (P11) and
P5. This proceeds through oxidative cleavage of the less hindered Beckmann rearrangement initiated by cyanuric chloride (Fig. 1c).
C–C bond of the hydroxy ketone, resulting in an intermediate con-
Finally, ring fusion to the eight-membered ring of P was achieved
taining an aldehyde and ester. Hemiacetal formation between the by intramolecular C–H insertion of a primary carbamate (Fig. 1d).
tertiary alcohol and aldehyde, followed by subsequent lactonization, Inspired by previous work on C–H amidation of the epi-mutilin
32
efficiently results in a ring rearrangement to form P5.
scaffold , ring fusion precursor P13 was synthesized by saponifi-
In addition to the direct formation of P1 from pleuromutilin, the cation of pleuromutilin followed by acylation and carbamylation
use of carbocation rearrangements is a useful strategy for inducing (Supplementary Scheme 4). Intramolecular C–H nitrene insertion
3
2
dramatic changes to the overlapping rings of pleuromutilin. In aim- was accomplished using a modified silver-catalysed methodology
ing to construct a subset of compounds in which the six-membered to provide the novel ring system found in carbamate P14. Exposure
26
ring was expanded, we identified alcohol P6, a common intermediate of this product to alkaline autoxidation conditions results in a
30,31
in the synthesis of pleuromutilin-derived antibiotics , as a useful formal ring expansion of the five-membered ring via enolate for-
substrate (Fig. 1b). P6 was generated via acid-catalysed isomeriza- mation, oxidative cleavage, hydride transfer and lactonization to
tion and a subsequent 1,5-hydride shift of pleuromutilin, as reported provide novel lactone P15 (Fig. 1d).
30
previously . Treatment of P6 with phosphorus pentachloride
Through the efforts reported here, 12 structurally complex
resulted in a novel expansion of the six-membered ring and elimina- compounds with novel ring systems were constructed from P, in
tion to form a new scaffold, P7, as a single isomer. Further modifi- addition to six compounds that had previously been reported; the
cation of P7 was accomplished by diastereoselective epoxidation of majority of these were synthesized on ≥25mg scale. In addition, the
the disubstituted olefin to afford novel scaffold P8. Elimination and compound collection derived from P that was screened (vide infra)
epoxide opening of P8 forms a new allylic alcohol P9 (Fig. 1b).
also included 11 compounds synthesized in our previously reported
Cleavage of the eight-membered ring of P has been reported transformation of pleuromutilin to P16 and carbocation rearrange-
24
33
to occur via a retro-Michael reaction . Indeed, oxidation of the ment to afford bridged oxafenestranes such as P17 (Fig. 1e) . The
3
secondary alcohol of pleuromutilin affords a 1,5-diketone that, fraction of sp -hybridized carbons (Fsp3), the number of stereo-
after retro-Michael ring cleavage with potassium hydroxide and genic centres and the ring complexity index were used as surrogates
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NaTuRe CHemISTRy
Articles
a
b
R1
R2
IC50 (µM)
Ph
P18 Cl
P19 Cl
P20 Cl
H
Me
Ac
Me
H
1.6 ± 0.1
1.9 ± 0.1
1.4 ± 0.5
>100
Ph
O
O
N
N
N
O
N
O
N
N
P21
P22
P23
H
F
I
Ph
O
O
34.0 ± 5.7
0.24 ± 0.002
>100
O
N
O
O
Cl
Cl
O
R1
O
HO
H
O
HO
HO
N
N
P24 AcO
H
CH2Cl2
82%
OR2
OH
OH
Cl
O
O
O
P25
Me
Me
>100
>100
P4
P18, Ferroptocide
Cl
IC50 (ES-2 cells) = 6.7 ± 0.4 µM
IC50 (ES-2 cells) = 1.6 ± 0.1 µM
O
P26
c
d
N
N
N
O
O
Ph
H
O
N
3
2
2
1
1
0
5
0
5
0
5
0
N
N
H
H
H
O
H
Cancer cell lines
Primary cancer cells
O
O
N
O
H
H
O
F
–B
O
O
F
HO
N
Cl
HO
Cl
HO
O
+
N
O
O
Cl
OH
O
OH
O
OH
O
P28
P29
4.0 ± 1.0 µM
d.r. = 1:1
P30
5.3 ± 0.1 µM
d.r. = 1:1
IC50 (ES-2 cells) 2.5 ± 0.4 µM
d.r. = 1:1
Hoechst
P30
Merged image
5
µm
5 µm
5 µm
Fig. 2 | Synthesis and evaluation of ferroptocide. a, Structure of P4, a hit compound in the cytotoxic phenotypic screen. Synthesis of lead compound P18
hereafter, ferroptocide). Below each compound is their respective 72ꢀhour IC50 value against ES-2 cells. Data represent meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological
(
replicates. b, Structure–activity relationship studies of P18 analogues; bioactivity is expressed as a 72ꢀhour IC50 value against the ES-2 cell line, as measured
by Alamar Blue fluorescence. Data represent meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological replicates. c, Ferroptocide displays broad activity in a 72ꢀhour cell viability
assay in immortalized cancer cells and in primary cells isolated from metastatic cancer patients. PPC, primary peritoneal carcinoma. Data represent
meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological replicates. d, Top, tool compounds P28, P29 and P30 retain biological activity in a 72ꢀhour cell viability assay in ES-2 cells.
Bottom, confocal microscopy images of ES-2 cells treated with fluorescent analogue P30 (1ꢀµM) for 15ꢀminutes show non-nuclear localization (green).
Nucleus was stained with Hoechst (blue), nꢀ=ꢀ3 biological replicates.
of complexity for the compounds synthesized from pleuromutilin, this compound is more potent than P4, with IC =1.6 μM against
5
0
and these values compare favourably to compounds in screening ES-2 cells. Further modification of the secondary alcohol and the
collections, as shown in the violin plots in Supplementary Fig. 1.
α-chloro ester of ferroptocide unveiled structural features impor-
tant for activity. Although methylation and acetylation of the sec-
Anticancer phenotypic screening and compound optimization. ondary alcohol of ferroptocide (P19 and P20, respectively) did
Compounds from P were evaluated in whole-cell assays for their not change the activity (Fig. 2b), replacement of the α-chloro ester
ability to rapidly kill cancer cell lines in culture, starting with the with acetate (P21) eliminated activity. To investigate other electro-
ES-2 (ovarian cancer) cell line. All compounds were assessed at philic groups the fluoro- (P22) and iodo- (P23) compounds were
1
2µM, with cell viability determined using the Alamar Blue via- synthesized. Although P22 had greatly diminished anticancer
bility assay. Compounds that elicited at least 50% cell death were activity, iodo analogues (such as P23) showed greater potency in
considered hit compounds and were then evaluated through full cells at the expense of biological selectivity (Supplementary Fig. 1),
dose–response curves. From these assessments, compound P4 was and because of this promiscuity such compounds were not pur-
identified as having promising activity, with rapid induction of cell sued further. Additional compounds with poorer leaving groups
death (a phenotype discussed further below) and a half-maximal such as α-acetate ester (P24), α,α-dichloro ester (P25) and furoic
inhibitory concentration (IC ) of 6.7μM in ES-2 cells (Fig. 2a); ester (P26) exhibited no anticancer activity (Fig. 2b). Lead com-
50
counter-screening revealed that this small molecule displayed no pound ferroptocide displays no antibacterial activity (minimum
−
1
signs of haemolytic activity (Supplementary Fig. 1).
inhibitory concentration, MIC >64 µg ml ) in Gram-positive
Modification of P4 through a [4+2] cycloaddition provided (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacte-
compound P18, hereafter referred to as ferroptocide (Fig. 2a); ria but has robust anticancer activity in a panel of cancer cell lines
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Articles
NaTuRe CHemISTRy
a
b
DMSO
30 min
Time to 50% cell death
20.28%
0
.98%
4.33%
1.84%
1
541B
Procaspase3
activators
PAC-1
Gemcitabine
Nucleoside
analogues
5
-Fluorouracil
MNNG
9
3.59%
74.95%
DNA
alkylators
Mitomycin C
Etoposide
Topoisomerase
inhibitors
Camptothecin
Cyclohexamide
Antimycin A
IB-DNQ
1.10%
2.92%
6
0 min
120 min
ROS
inducing agents
11.85%
29.43%
1.26%
1.61%
95.39%
Rotenone
Staurosporine
Taxol
Kinase inhibitor
Microtubule stabilizer
Proteasome
Bortezomib
Raptinal
5
4.42%
Apoptosis inducer
Ferroptocide
4
.30%
1.73%
0
4
8
12 16 20 24 28 32 36 40 44 >48
Time (h)
Annexin V
c
d
Control
Ferroptocide (10 µM)
STS (10 µM)
ES-2 cells
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
No Q-VD-OPh
Q-VD-OPh
***
NS
1
µm
1 µm
1 µm
NS
NS
µM)
µM)
µM)
µM)
DMSO
Raptinal (5
Ferroptocide (5
Ferroptocide (10 Ferroptocide (25
100 nm
100 nm
100 nm
e
Hoechst
P30
f
7
5
3
1
3
5
7
8
0
ES-2 cells
DMSO
TBHP (100 µM)
Etoposide (100 µM)
Ferroptocide (5 µM)
Ferroptocide (10 µM)
Ferroptocide (25 µM)
5
µm
5 µm
2
0
2
3
4
5
–
10 –10 10
10
10
10
Carboxy-H DCFDA fluorescence
2
5
µm
5 µm
Mitotracker
Merged image
Fig. 3 | Ferroptocide induces rapid non-apoptotic cell death. a, Speed of death of cells treated with ferroptocide versus 16 other anticancer compounds
in ES-2 cells (all tested at 10ꢀµM). Cell viability was assessed by AV/PI analysis. Representative data are taken from full time-course data (three biological
replicates) in Supplementary Fig. 8. b, Time-course analysis of ES-2 cell viability following treatment with ferroptocide (10ꢀµM) indicates a non-apoptotic
mode of cell death. AV/PI graphs are representative of three biological replicates. c, Effect of pretreatment with Q-VD-OPh (25ꢀµM) for 2ꢀhours followed by
dose–response treatment with ferroptocide or positive control Raptinal (5ꢀµM) for 8.5ꢀhours in ES-2 cells. Data are plotted as meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological
replicates. Two-sided t-test, ***Pꢀ=ꢀ0.0003, NS Pꢀ>ꢀ0.05. d, TEM images of ES-2 cells treated with DMSO (left), ferroptocide (10ꢀµM, centre) or STS
(
10ꢀµM, right) for 30ꢀminutes. The images show lack of apoptotic morphological features and swelling of mitochondria upon ferroptocide treatment (red
arrows) versus controls. TEM data are representative images of three technical replicates. e, Co-localization analysis with mitochondria. ES-2 cells were
stained with MitoTracker Red (100ꢀnM) followed by 30ꢀmin treatment with fluorescent analogue P30 (10ꢀµM). Nucleus was stained with Hoechst. Yellow
dots indicate P30 (green) on the mitochondria (red) in merged images, nꢀ=ꢀ3 independent experiments. f, Ferroptocide induces dose-dependent ROS
generation within 1ꢀhour, similar to positive control TBHP in ES-2 cells (and also in HCT 116 cells; see Supplementary Fig. 2). DMSO and etoposide were
included as negative controls. Data are representative of three independent experiments.
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NaTuRe CHemISTRy
Articles
a
b
ES-2 cells
ES-2 cells
ES-2 cells
A549 cells
1
00
No trolox
Trolox
8
4
3
9
0
0
****
****
*
**
8
DMSO
6
70
60
50
DMSO + DFO
Ferroptocide (10 µM)
*
*
4
2
40
30
20
10
0
Ferroptocide + DFO
RSL3 (10 µM)
21
RSL3 + DFO
0
1
2
3
4
5
–
10 10
10
10
10
C11-BODIPY fluorescence
c
d
No ferrostatin-1
Ferrostatin-1
No DFO
DFO
ES-2 cells
1
00
1
00
*
**
*
***
*
***
90
****
9
8
7
6
5
4
3
2
1
0
****
****
80
70
60
**
0
0
0
0
0
0
0
0
0
***
50
40
30
20
10
0
NS
NS
e
HCT 116 cells
1
00
100
90
80
70
60
50
40
30
20
10
0
Ferroptocide (10
RSL3 (10 M)
Erastin (10 M)
µM)
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
µ
µ
Ferroptocide (10
RSL3 (10 M)
Erastin (10 M)
µM)
µ
µ
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
Fig. 4 | Ferroptocide kills cancer cells through ferroptosis. a, Ability of iron chelator DFO to prevent ferroptosis upon treatment with ferroptocide or
positive control RSL3 for 1ꢀhour in ES-2 cells (C11-BODIPY probe, lipid ROS). Data are representative of three independent biological experiments.
b, Lipophilic antioxidant trolox (250ꢀµM) rescues ES-2 cells from ferroptocide-induced cytotoxicity after a 14ꢀhour incubation. c, Ability of ferroptosis inhibitor
ferrostatin-1 (2ꢀµM) to protect cells against ferroptocide treatment after 14ꢀhours in ES-2 cells. d, Effect of DFO (100ꢀµM) on viability of ES-2 cells after a
1
4ꢀhour incubation with ferroptocide and erastin (positive control). e, Comparison of speed of cell death with ferroptocide, RSL3 and erastin, each at 10ꢀµM
in HCT 116 and A549 cells (two K-RAS mutant cancer cell lines), respectively. In b–e, cell viability was determined with AV/PI staining. Data are plotted as
−5
meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological replicates. Two-sided t-test, ****Pꢀ<ꢀ0.0001, ***0.0001ꢀ≤ꢀPꢀ<ꢀ0.001, **0.001ꢀ≤ꢀPꢀ<ꢀ0.01, NS Pꢀ>ꢀ0.05 (P values were 1.1ꢀ×ꢀ10 ,
−6
−8
−6
−7
−8
−9
1.8ꢀ×ꢀ10 , 0.0001, 0.009 for trolox; 3.3×10 , 6.5×10 , 1.7×10 , 0.0008 for ferrostatin-1; 0.0002, 3.3×10 , 4.5×10 , 0.003 for DFO from left to right).
and, notably, primary cancer cells freshly isolated from tumour and experimental chemotherapeutics such as cisplatin, 5-fluoro-
tissues of 15 different patients with diverse metastatic cancers uracil (5-FU), etoposide and procaspase-activating compound 1
(
Fig. 2c). Ferroptocide kills these cancer cells better than approved (PAC-1) (Supplementary Fig. 1).
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Articles
NaTuRe CHemISTRy
0
µM
20 µM
Ferroptocide
a
b
P30 (1 µM)
–
+
+
+
+
+
+
+
+
+
P30 (µM)
0
0.1 0.5
1
M (kDa)
M
(kDa)
w
w
7
5
5
0
75
A A′
50
3
7
3
2
7
5
B
B
25
C
D
10
10
D
c
d
Ferroptocide (20 µM)
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
Ferroptocide (20 µM)
P30 (1 µM)
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
P30 (1 µM)
Mw (kDa)
M
(kDa)
w
7
5
3
5
0
7
7
5
3
5
0
7
B
D
B
2
5
2
1
5
0
1
0
D
HCT 116
ES-2
U937
N
MIA PaCa-2 BT-549
T47D MDA MB-231
Not significant
Lung
Ovarian
PPC
e
f
g
O
H
O
Significant
Mw
(kDa)
Protein
Fold change
H
Fold change >3
TXNRD1
TXNRD3
TXNRD1
76.4
7.4
4.9
O
KEAP1
70.8
69.7
61.1
TXN PTGES2
Cl
HO
P29
Whole cells
O
PGLS
KEAP1
PDP1
3.1
3.7
TXNRD3
GSTO1
OH
P < 0.05
PDP1
O
0.05
0.25
TXNRD2
PTGES2
PGLS
56.4
41.9
27.5
27.5
2
1
. Pretreat with ferroptocide/DMSO
. Treat with alkyne probe
TXNRD2
2
18
2.7
3.6
3
4
5
. ‘Click’
. Enrich
0
1
.50
.00
. On-bead tryptic digest
GSTO1
TXN
11.7
11.1
Potential
target
Discard
target
0.5
1.0
2.0
4.0
8.0
16.0
32.0
Fold change
m/z
m/z
Fig. 5 | Ferroptocide selectively and covalently modifies its target in cells. a, Proteomic profile for fluorescent analogue P30 in HCT 116 cells after a
0ꢀminute treatment reveals labelling of five main bands (nꢀ=ꢀ3 biological replicates). (Note that bands A and Aʹ often appear as one band). Coomassie
staining of the gel demonstrates equal loading (Supplementary Fig. 9a). b, Competitive profiling of the proteomic reactivity of P30 with ferroptocide. HCT
6
1
16 cells were pretreated with DMSO or various concentrations of ferroptocide (30ꢀmin) followed by treatment with P30 (1ꢀµM, 30ꢀmin) and then in-gel
fluorescence analysis (nꢀ=ꢀ3 biological replicates). Coomassie staining of the gel demonstrates equal loading (Supplementary Fig. 9b). c, Ferroptocide
covalently modifies the same target(s) in multiple cell lines. Competition experiments were performed by treatment of cells with DMSO or ferroptocide
(
20ꢀµM, 30ꢀminutes) followed by P30 incubation (1ꢀµM, 30ꢀminutes) and then analysed using an in-gel fluorescence assay. Images are representative of
three biological replicates. Coomassie staining of gels demonstrates equal loading (Supplementary Fig. 9c). d, Ferroptocide causes the same proteomic
competitive profile in primary cells isolated from metastatic cancer patient samples. These cells were pretreated with DMSO or ferroptocide (20ꢀµM,
3
0ꢀminutes), followed by P30 incubation (1ꢀµM, 30ꢀminutes) and in-gel fluorescence analysis. Images are representative of two biological replicates. PPC,
primary peritoneal carcinomatosis. Coomassie staining of gels demonstrates equal loading (Supplementary Fig. 9d). e, Schematic of the biotin–streptavidin
pulldown method: treatment of HCT 116 cells with ferroptocide (20ꢀµM, 30ꢀminutes) and P29 (20ꢀµM, 60ꢀminutes) was followed by CuAAC reaction with
biotin-azide and enrichment with streptavidin magnetic beads. On-bead trypsin digestion coupled to LC/LC–MS/MS provided a list of over 300 targets
(
Supplementary Data 2). f, Enrichment of proteins based on Pꢀ<0.05 and fold changeꢀ>3 in HCT 116 cells, nꢀ=ꢀ2 independent experiments, two-sided
student t-test. TXN was a top target candidate. g, Proteins identified for follow-up characterization based on shared enrichment in both HCT 116 and ES-2
cell lines, as well as molecular weights matching the bands observed by gel.
Additional synthesis and evaluation revealed that the N–N moi- of alkyne tool compound P29 (Fig. 2d), which was subjected to
ety in ferroptocide could be changed to C–C (P28, Fig. 2d), with a 1,3-dipolar cycloaddition resulting in fluorescent compound
minimal loss in activity. This discovery allowed for the construction P30. Both P29 and P30 retained anticancer activity (Fig. 2d), and
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a
b
c
100 ES-2 cells
TXN-GFP
WT
NS
90
80
70
60
50
40
30
20
10
0
Ferroptocide (20
P29 (20
µ
µM)
M
)
–
–
–
+
+
+
–
–
–
+
+
+
*
BPD
Input
*
*
P29 (20 µM)
–
+
–
+
Trx
37 kDa
Actin
Ferroptocide
20 M)
PMX464
PX-12
(50 µM)
(
µ
(50
µM)
d
WT
C32S
C35S
Control
WT
C62S
C69S
C73S
Ferroptocide (20
P29 (5
µ
M
)
–
–
–
+
+
+
–
–
–
+
+
+
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
–
–
–
+
+
+
µ
M
)
–
3
7 kDa
e
f
C32S
1
00
HCT 116 cells
siRNA Neg
siRNA TXN
C73S
90
C62S
Active site
C35S
NS
80
70
*
60
50
40
30
20
10
0
C69S
*
*
*
**
NS
*
*
*
*
Fig. 6 | Ferroptocide modulates active-site cysteines of thioredoxin. a, Immunoblot of thioredoxin pulldown upon treatment of HCT 116 cells with DMSO
or P29 (20ꢀµM, 60ꢀminutes) followed by CuAAC reaction with biotin-azide and streptavidin enrichment: BPD (biotin pulldown) and input (soluble cell
lysate). Images are representative of three biological experiments. b, Effect of ferroptocide (20ꢀµM) and known inhibitors PMX464 and PX-12 (50ꢀµM) on
thioredoxin activity in ES-2 cells after 30ꢀminute incubation. Data are presented as meanꢀ±ꢀs.e.m; two-sided student t-test, P values are relative to DMSO
control (0.003 and 0.03 from left to right); **0.001ꢀ≤ꢀPꢀ<ꢀ0.01, *0.01ꢀ≤ꢀPꢀ<ꢀ0.05, NS Pꢀ>ꢀ0.05, nꢀ=ꢀ3 independent experiments. c, Competition profile of
thioredoxin labelling by probe P29 (20ꢀµM, 60ꢀminutes) upon pretreatment with DMSO or ferroptocide (20ꢀµM, 30ꢀminutes) followed by CuAAC with
Cy3 azide in HCT 116 cells overexpressing TXN-GFP plasmid versus non-transfected (wild type, WT) cells, Cy3 channel. The red box indicates competition
of the band of interest. Representative in-gel fluorescence images of nꢀ=ꢀ3 biological replicates. Coomassie staining of gel demonstrates equal loading
(
Supplementary Fig. 10). d, Identification of ferroptocide labelling sites on thioredoxin. In-gel fluorescence scanning of HCT 116 cells overexpressing each
cysteine-mutated thioredoxin. Cells were pretreated with DMSO or ferroptocide (20ꢀµM, 30ꢀminutes) followed by incubation with P29 probe (5ꢀµM,
0ꢀminutes) and then CuAAC reaction with Cy3 azide. The serine mutations of the active-site cysteines 32, 35 and cysteine 73 diminished compound
6
labelling. Data are representative of three biological replicates. Coomassie staining of gels demonstrates equal loading (Supplementary Fig. 11).
e, Crystal structure of thioredoxin with cysteine residues coloured in red (PIB:1ERT). f, siRNA of thioredoxin in HCT 116 cells (48ꢀhours) sensitizes them
to ferroptocide treatment (10ꢀµM) but not Raptinal (10ꢀµM) at the indicated time points. Note, siRNA of TXN at 48ꢀhours is not toxic to cells. Cell viability
was determined with AV/PI staining. Data are plotted as meanꢀ±ꢀs.e.m., nꢀ=ꢀ3 biological replicates. Two-sided student t-test, ***0.0001ꢀ≤ꢀPꢀ<ꢀ0.001,
*
*0.001ꢀ≤ꢀPꢀ<ꢀ0.01, NS Pꢀ>ꢀ0.05 (P values were 0.04, 0.004, 0.0006, 0.03, 0.04 and 0.006 from left to right).
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1
,000
1,000
800
600
400
200
0
Vehicle
Vehicle
–1
–1
Ferroptocide (50 mg kg
)
Ferroptocide (50 mg kg )
8
6
4
2
00
00
00
00
0
*
*
*
Balb/c mice
SCID mice
8
11
14
17
20
23
8
11
14
17
20
23
Days after 4T1 cells inoculation
Days after 4T1 cells inoculation
Fig. 7 | Ferroptocide modulates the immune system. Ferroptocide inhibits subcutaneous 4T1 tumour growth in immunocompetent Balb/c mice (left)
−
1
but not in immunodeficient SCID mice (right) as measured by tumour volume. Ferroptocide was administered intraperitoneally at 50ꢀmgꢀkg , twice a
week, for five doses (nꢀ=ꢀ7 mice per group). Data represent the meanꢀ±ꢀs.e.m. Two-sided student t-test, P values are relative to vehicle control; **Pꢀ<ꢀ0.01,
*
0.01ꢀ≤ꢀPꢀ<ꢀ0.05 (0.009 and 0.04 from left to right).
P30 was used to report on subcellular localization. As shown by confocal microscopy studies supported such findings as the fluo-
confocal microscopy, P30 localizes to the cytoplasm in ES-2 cells rescent analogue P30 was found to co-localize with the Mitotracker
(
Fig. 2d), and this staining is competed away by pretreatment with dye in cells (Fig. 3e) while the BODIPY azide dye alone did not
ferroptocide (Supplementary Fig. 1). Importantly, installation of (Supplementary Fig. 2), thus suggesting a mitochondria-based activity
an α-chloro ester on pleuromutilin itself (P27), and other scaffolds of ferroptocide. Given the importance of ROS generation in mitochon-
such as lovastatin (L1) and quinine (QQ1), resulted in non-com- dria, ROS levels were monitored upon compound treatment using a
peting compounds in this localization experiment (Supplementary ROS probe, carboxy-H DCFDA. Dose-dependent ROS production
2
Fig. 1), demonstrating that the anticancer activity of ferroptocide was observed in ES-2 (Fig. 3f) and HCT 116 cells (Supplementary
is not attributed solely to the presence of the electrophilic func- Fig. 2) treated with ferroptocide, similar to the positive control, tert-
tional group.
butyl hydroperoxide (TBHP). Furthermore, treatment of ES-2 cells
with ferroptocide results in an increase of mitochondrial ROS simi-
39
Ferroptocide induces non-apoptotic cell death. To gain insights larly to treatment with positive controls IB-DNQ and rotenone
into the mode of action, the speed of cell death of ferroptocide was (Supplementary Fig. 2). Collectively, these data support a disruptive
compared to other approved chemotherapeutics and tool com- role of ferroptocide on mitochondrial activity.
pounds with well-defined mechanisms, including procaspase-3 acti-
3
4
vators (PAC-1, 1541B) , nucleoside analogues (gemcitabine, 5-FU), Ferroptocide is a pro-ferroptotic agent. One non-apoptotic mode
DNA alkylators (MNNG, mitomycin C), topoisomerase inhibitors of cell death that depends on the production of lethal levels of
(
(
etoposide, camptothecin, cycloheximide), reactive oxygen species iron-dependent lipid ROS is ferroptosis, a regulated process with
3
5
4
0
–
4
4
ROS) inducing agents (anitimycin A, IB-DNQ , rotenone), broad- distinct morphological, biochemical and genetic characteristics
spectrum kinase inhibitor (staurosporine), microtubule stabilizer that shares similar features with another non-apoptotic form of cell
45–48
(
taxol), proteasome inhibitor (bortezomib) and a rapid apoptosis- death, oxytosis .The hallmarks of ferroptosis include generation
36
inducing agent (Raptinal) . The cell death induced by ferropto- of lipid hydroperoxides and cytoprotection by lipophilic antioxi-
cide was rapid in multiple cell lines of diverse cancer types, with a dants (trolox, butylated hydroxyltoluene (BHT)), ferroptosis inhibi-
time to 50% cell death of 1h in ES-2 (Fig. 3a), 1.5h in Mia PaCa-2 tors (ferrostatin-1, liproxstatin) and iron chelators (deferoxamine
40
and 7h in HCT 116 cells (Supplementary Fig. 2). As the speed of (DFO), ciclopirox olamine (CPX)) . Cellular effects of ferroptocide-
cell death induced by ferroptocide was faster than the most rapid induced ROS were investigated using a C11-BODIPY probe that
41
proapoptotic agent known (Raptinal), it was suspected to induce responds to lipid peroxidation . Ferroptocide induces lipid ROS
non-apoptotic cell death.
in ES-2 (Fig. 4a), HCT 116 and 4T1 cells (Supplementary Fig. 3),
Time-course analysis of cells treated with ferroptocide fol- similar to the known ferroptosis inducer (1S,3R)-RSL3 (hereafter
49
lowed by annexin-V/propidium iodide (AV/PI) staining indeed RSL3) and/or TBHP; DFO pretreatment of these cells abolishes
suggested a non-apoptotic mode of cell death (Fig. 3b), as did each compound’s activity. Given that generation of continuous lipid
40,41
experiments showing that the pan-caspase inhibitor Q-VD-OPh ROS is a functional requirement of ferroptosis , additional exper-
does not protect against ferroptocide-induced cell death in ES-2 iments were conducted to elucidate if ferroptosis was triggered
(
Fig. 3c) and HCT 116 cells (Supplementary Fig 2). Cleavage of by ferroptocide. Protection studies were conducted with trolox,
PARP-1 in ferroptocide-treated ES-2 or HCT 116 cells was not ferrostatin-1 and DFO, and all these inhibitors significantly pro-
observed (Supplementary Fig. 2). As a further confirmation, cell tected against ferroptocide-induced cell death in ES-2 (Fig. 4b–d),
morphological changes induced by ferroptocide were examined HCT 116, 4T1 and A549 cancer cells (Supplementary Fig. 3).
using transmission electron microscopy (TEM). Cells treated with Additionally, these inhibitors rescued cells from TBHP treatment
ferroptocide exhibit none of the apoptotic characteristics such as (Fig. 4b) and the known ferroptosis inducers erastin (Fig. 4c,d)
membrane blebbing and chromatin condensation (Fig. 3d; see stau- and RSL3 (Supplementary Fig. 3), respectively, while they showed
rosporine (STS) control). Together, these data indicate that ferrop- no protection against Raptinal, an apoptosis-inducing agent.
tocide induces rapid, non-apoptotic cell death. As compounds with Treatment of HCT 116 and A549 cells with the antioxidant N-acteyl
such a mode of cell death can have unique properties and advan- cysteine (NAC-1) resulted in protection from ferroptocide- and
3
7,38
tages in vivo , further elucidation of the mechanism of cell death TBHP-induced cell death, but not Raptinal (Supplementary Fig. 3).
of ferroptocide was of interest.
Together, these studies indicate that iron-dependent accumulation
Further analysis of the TEM images revealed mitochondrial of lipid peroxidation (ferroptosis) upon ferroptocide treatment is
swelling as early as 30min after ferroptocide treatment. Subsequent the cause of cell death.
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Erastin and RSL3 were originally discovered as small molecules spectral counting (Fig. 5f). High-affinity targets from the HCT
41
with RAS-selective lethality . Monitoring of speed of cell death of 116 cell line were then compared to targets identified in ES-2 cells
ferroptocide versus erastin and RSL3 in HCT 116 and A549 cells (Supplementary Data 2). Based on shared enrichment in both cell
(
which contain mutant oncogenic K-RAS) demonstrates that ferrop- lines, as well as molecular weights matching the gel bands, nine
tocide is a fast-acting, robust pro-ferroptotic agent inducing more proteins were selected for follow-up characterization (Fig. 5g).
quantitative cell death than the other tool compounds (Fig. 4e). Importantly, GPX4 protein was not identified as a target of inter-
Additionally, treatment of HCT 116 cells with the same concentra- est for ferroptocide, with low spectral counts below the cutoff for
tion of these compounds results in generation of similar levels of significance.
lipid ROS upon ferroptocide and RSL3 treatment and a larger quan-
To discriminate between on- and off-cytotoxicity-related targets,
tity compared to erastin treatment, suggesting a rapid onset of lipid siRNA and CRISPR–Cas9 strategies were used. Following successful
peroxidation for ferroptocide and RSL3 (Supplementary Fig. 3). siRNA knockdown of KEAP1 and GSTO1, an assessment was made
Given that RSL3 is a covalent inhibitor of a central regulator of of how changes in protein expression affected band labelling in gels.
40,45
ferroptosis, GPX4 , we monitored if ferroptocide modulates the Comparison of cells with knockdown targets and wild-type cells
activity of GPX4 in cells. As shown in Supplementary Fig. 3, treat- indicated no change in in-gel fluorescence (Supplementary Fig 5),
ment of ES-2 cells with ferroptocide did not result in GPX4 inhibi- suggesting that KEAP1 and GSTO1 are off-pathway targets of fer-
tion (in contrast to the positive control RSL3), suggesting a different roptocide. CRISPR–Cas9 technology was then used to rapidly
target for ferroptocide.
investigate the remaining targets. We were able to successfully gen-
Further experiments were conducted to monitor the effect of erate isogenic cell line pairs for six knockout targets, with one other
ferroptocide at the transcript level. RNA–seq data of ferroptocide- target being lethal. Knockout of six targets did not diminish label-
treated cells revealed that 35 of 40 genes involved in ferroptosis are ling of any of the fluorescent bands, indicating that these proteins
modulated with false discovery rate (FDR) scores of ≤0.05 upon (PTGES2, PGLS, TXNRD1, TXNRD2, TXNRD3 and PDP1) were
6
h treatment (Supplementary Fig. 3). This time point was selected not the targets of interest (Supplementary Fig. 5), while the lethal
to capture the primary mechanisms of the compound of interest target corresponded to that of thioredoxin protein.
50
on viable cells (Supplementary Fig. 3), as described previously .
Specific genes such as GCLC (3.5-fold), GCLM (4.9-fold), SLC7A11 plays a key role in the thioredoxin antioxidant system (compris-
Thioredoxin (TXN) is a 12kDa ubiquitous oxidoreductase that
(
8.1-fold) and CHAC1 (9.8-fold), known to be upregulated in fer- ing thioredoxin, NADPH and thioredoxin reductase). Thioredoxin
40,51
roptosis , and endoplasmic reticulum stress (ATF3 11.5-fold, contains five cysteines and uses active-site cysteines (C32 and C35)
DDIT3 22.5-fold, DDIT4 11.3-fold) were significantly upregu- to reduce the disulfide bonds of many protein partners such as tran-
lated after ferroptocide treatment, similar to RNA–seq reports for scription factors (NF-κB, AP-1, Ref-1), ribonucleotide reductases,
51
erastin in HT-1080 cells . Pathways affected by oxidative stress peroxiredoxins and glutathione peroxidases, as well as scavenging
−
10
53,54
such as Keap1-Nrf2 (P=7.8×10 ), unfolded protein response of ROS . Treatment of HCT 116 cells with P29, coupled to bio-
−
8
(
(
P=3.6×10 ), protein processing in endoplasmic reticulum tin–streptavidin enrichment followed by immunoblotting, yielded a
−10
P=2.4×10 ) and others were also modified upon compound band present only in compound-treated sample (Fig. 6a), suggesting
treatment (Supplementary Fig. 3 and Supplementary Data 1). These that ferroptocide covalently modifies thioredoxin. A thioredoxin
transcription profiles provide further support that ferroptocide activity assay was then used to assess the ability of ferroptocide to
induces oxidative stress and ferroptosis.
inhibit thioredoxin activity in cell lysate. Ferroptocide significantly
reduced the activity of thioredoxin (Trx) in ES-2 cells to a greater
Ferroptocide covalently modifies thioredoxin. Structure–activ- extent than the two known inhibitors of thioredoxin (PMX464 and
55–57
ity relationship trends reveal that ferroptocide bioactivity depends PX-12)
(Fig. 6b). Dose–response analysis confirmed that ferrop-
on the presence of the electrophilic α-chloroester (Fig. 2), suggest- tocide is also a more potent thioredoxin inhibitor than PMX464 and
ing covalent modification of its target. In vitro studies indicate that PX-12 in a biochemical (in vitro) assay (Supplementary Fig. 5).
ferroptocide reacts slowly with excess glutathione (67% compound
To further assess the effect of ferroptocide on thioredoxin,
remaining after 2h) compared to the rapid reaction of the promis- thioredoxin fused to GFP (TXN-GFP, 37kDa) was overexpressed
cuous iodo analogue P23 (Supplementary Fig. 4). To assess covalent in HCT 116 cells (Supplementary Fig. 5). Treatment of these cells
modification in cells, in-gel fluorescence studies were performed in with P29, followed by bioconjugation of the orthogonal fluorophore
conjunction with competition studies. Treatment of HCT 116 cells of Cy3, afforded the expected band at 37kDa (Fig. 6c), which was
with increasing concentrations of fluorescent analogue P30 resulted competed by ferroptocide. To identify the sites of modification of
in labelling of five main bands (Fig. 5a). Pretreatment of cells with thioredoxin by ferroptocide, site-directed mutagenesis introduced
various concentrations of ferroptocide, followed by treatment with serine mutants of each of the five cysteines of TXN-GFP. The ability
P30, resulted in dose-dependent competition, primarily of two of ferroptocide to covalently modify these mutant proteins (C32S,
bands, in the in-gel fluorescence assay (bands B and D in Fig. 5b). C35S, C62S, C69S and C73S, Supplementary Fig. 5) was assessed
A similar labelling and competition pattern was observed in mul- in a fluorescent band labelling experiment. As shown in Fig. 6d, the
tiple cancer cell lines, including HCT 116, ES-2, U937, MIA PaCa- 37kDa band is not present in the C32S and C35S mutants and has
2
, BT-549, T47D, MDA-MB-231 (Fig. 5c) and primary cancer cells reduced labelling in the C73S mutant, suggesting that ferroptocide
from patients (Fig. 5d), suggesting modulation of the same targets is modifying the active-site cysteines and the adjacent cysteine 73
in immortalized and in primary cancer cells. of thioredoxin as shown in the crystal structure (Fig. 6e). Taken
In an effort to identify the labelled protein(s), a biotin–strepta- together, these studies demonstrate that treatment with ferroptocide
52
vidin pulldown (schematic in Fig. 5e) was performed with alkyne modifies critical residues needed for interaction of thioredoxin with
P29. Briefly, HCT 116 cells were pre-treated with ferroptocide or its binding partners, and thus inhibiting its activity in cells. This
DMSO, followed by treatment with P29. Upon incorporation of a inhibition presumably causes the observed phenotype of rapid fer-
biotin, P29-labelled proteins were enriched on streptavidin beads, roptotic cell death. Given that thioredoxin is a key component of a
subjected to an on-bead trypsin digestion and subsequent liquid major antioxidant system, it is possible that its modulation renders
chromatography-mass spectrometry (LC/LC–MS/MS) analysis. cells susceptible to the oxidative stress that causes lipid peroxidation
Protein identities were determined by database searches using the and other imbalances in cellular processes, which eventually lead
SEQUEST algorithm. Relative quantitation of proteins enriched to ferroptotic cell death; other thioredoxin inhibitors have not been
in ferroptocide and DMSO pretreated samples was achieved by reported to induce ferroptosis.
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General and lipid ROS levels were monitored upon genetic
Here, we report application of the CtD strategy to the natural
knockdown of thioredoxin in HCT 116 cells. Knockdown of thio- product of pleuromutilin, which provided a set of 29 structurally
redoxin (siTXN) resulted in massive generation of general ROS diverse and highly complex compounds. This set was then subjected
and lipid ROS within 72h (Supplementary Fig. 6), consistent with to a phenotypic screen that allowed discovery of ferroptocide, which
induction of ferroptosis. Pretreatment of HCT 116 cells with fer- induces rapid ferroptotic death in immortalized cancer cell lines and
roptosis inhibitors of trolox and ferrostatin-1 did not protect against primary cancer cells from patients. Cell culture studies demonstrate
siTXN, however, probably due to high ROS levels and long incuba- that ferroptocide-treated cells generate ROS and lipid peroxidation,
tion times required for sufficient knockdown of thioredoxin; pre- which result in inevitable cell death, an effect that can be prevented
treatment with DFO impaired cell viability even in the control HCT by pretreatment with known inhibitors of ferroptosis (trolox, fer-
1
16 cells transfected with scrambled RNA (siNeg, Supplementary rostatin-1 and DFO). Depletion of the glutathione antioxidant sys-
Fig. 6). To determine if thioredoxin inhibitors cause ferroptotic cell tem and pharmacological inhibition or degradation of glutathione
death, protection studies with ferroptosis inhibitors were conducted peroxidase 4 (GPX4) are the main known systems that control fer-
40,41,43,44,51,64
in two cell lines; minimal protection was observed in ES-2 cells roptosis
while A549 displayed no change in cell death compared to untreated roptocide targets a different antioxidant system (thioredoxin) to
cells (Supplementary Fig. 6). These results are unsurprising given induce ferroptosis; it is likely that inhibition of thioredoxin causes
. In contrast, the collected data suggest that fer-
55,58
59,60
that PMX464 and PX-12 are imperfect tool compounds, sus- a drastic imbalance in the ROS levels, overwhelms cellular antioxi-
pected to engage multiple molecular targets in cells. The siRNA dant responses (as seen at the transcript level) and causes ferropto-
knockdown of thioredoxin sensitizes HCT116 cells to ferropto- sis. This hypothesis is supported by genetic knockdown studies of
cide but not Raptinal treatment in a time-course study (Fig. 6f and thioredoxin, which lead to accumulation of large amounts of ROS,
Supplementary Fig. 6), which is further suggestive of the impor- lipid ROS and sensitization of siTXN cells to ferroptocide treatment.
tance of thioredoxin in ferroptocide-induced cell death. Together, Nevertheless, further mechanistic details of the thioredoxin–ferrop-
these data support a role of thioredoxin in ferroptocide-induced cell tosis link remain to be elucidated, and the possibility that there are
death and ferroptosis.
alternative targets important for the pro-ferroptotic action of fer-
roptocide cannot be ruled out.
Ferroptocide is an immunostimulatory compound. The non-
Head-to-head comparisons of ferroptocide to erastin and RSL3
apoptotic nature of ferroptocide inspired preliminary explora- suggest that ferroptocide may have advantages, especially for appli-
tion of its ability to modulate the immune system. Non-apoptotic cations requiring induction of rapid and/or quantitative ferroptotic
compounds are attractive anticancer agents, as they can potentially cell death. Furthermore, as ferroptocide induces a regulated, non-
3
7
elicit an immune response . Ferroptocide displays some activity apoptotic mode of cell death, this compound (and possibly other
in non-cancerous breast (MCF10A) and human skin fibroblast pro-ferroptotic agents) has the potential to synergize with the
(
HFF-1) cells (IC of 3.1 and 4.1 µM, respectively), but no haemo- immune system for the treatment of cancer. Ferroptocide represents
5
0
lytic activity, so it is a favourable tool compound to assess in vivo. a distinct class of ferroptosis inducers and will be an important tool
We investigated the role of the immune system by assessing the compound for further studies of ferroptosis.
efficacy of ferroptocide in a subcutaneous murine model of 4T1
triple negative breast cancer cells in immunocompetent (Balb/c) Reporting Summary. Further information on research design
compared to immunocompromised (severe combined immuno- is available in the Nature Research Reporting Summary linked to
deficiency, SCID) mice. Measurements of tumour volume indi- this article.
cated a 40% tumour growth retardation in compound-treated
Balb/c compared to vehicle-treated mice, with activity probably Data availability
limited by poor compound pharmacokinetics in mice (Fig. 7 and The data supporting the findings of this study are available within
Supplementary Fig. 7). The lack of activity in immunocompro- the paper and Supplementary Information and are available from
mised mice suggests that T and B cells play a role in the activity of the corresponding author upon request. The mass spectrometry
ferroptocide in vivo.
proteomics data have been deposited to the ProteomeXchange
Consortium via the PRIDE partner repository with the data set
identifier PXD012805 (www.ebi.ac.uk/pride/archive/projects/
Conclusions
Small molecules are powerful tools for investigating protein func- PXD012805). The RNA sequencing data have been deposited to
tion and cell death mechanisms. Staurosporine and more recently the GEO repository with accession number GSE126868 (www.
36
Raptinal are commonly used to predictably and rapidly induce ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126868). The X-ray
apoptotic cell death and enable the study of its mechanisms and crystallography data have been deposited in the Cambridge
protein regulators. Selective inhibitors of cell death processes are Crystallographic Data Centre (CCDC) using the following identi-
also extremely valuable, with z-VAD-fmk and Q-VD-OPh widely fiers (www. ccdc.cam.ac.uk/structures/): 1851845 (compound P1)
used to inhibit apoptosis, and necrostatin-1 and ferrostatin-1 used and 1849494 (Ferroptocide).
61
to inhibit necrosis and ferroptosis, respectively .
In contrast to the variety of tool compounds available to induce Received: 2 July 2018; Accepted: 25 March 2019;
Published: xx xx xxxx
apoptosis, there are comparatively fewer that can be used to induce
62
ferroptosis, another regulated form of cell death . Erastin and
RSL3 are the first reported inducers of ferroptosis, followed by
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Acknowledgements
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NAꢁꢂRE CHEMꢃSꢁRY | www.nature.com/naturechemistry

Articles
NaTuRe CHemISTRy
and T. Woods for X-ray analysis of compounds, L. Dirikolu for calculation of
pharmacokinetic (PK) parameters, B. Drown for chemoinformatic analysis of
compounds, M.E. Vinyard and A. Sowers for synthetic assistance and S. Tasker for many
helpful discussions.
Competing interests
The University of Illinois has filed patents on some compounds described in this
manuscript.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-019-0261-6.
Author contributions
P.J.H., E.L. and R.W.H. conceived this study. R.W.H. designed and synthesized
all compounds. E.L. designed and performed all biological experiments and
analysed data. H.Y.L. performed the mouse model studies. S.E.M. synthesized the
lead compound for the mouse model. L.A.C. and E.W. collected and analysed the
LC/LC–MS/MS data. P.J.H. supervised this research. P.J.H. and E.L. wrote this
manuscript with the assistance of R.W.H. All authors have given their approval of the
final version of the manuscript.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to P.J.H.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2019
NAꢁꢂRE CHEMꢃSꢁRY | www.nature.com/naturechemistry

Corresponding author(s): Paul J. Hergenrother
Last updated by author(s): Feb 21, 2019
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