Activity-based protein profiling reveals GSTO1 as the covalent target of piperlongumine and a promising target for combination therapy for cancer

Article abstract of DOI:10.1039/c9cc00917e

Through systematic target identification for piperlongumine, a cancer-selective killing molecule, we identified GSTO1 as its major covalent target for cancer cell death induction. We also reveal that GSTO1 inhibition is a promising combination strategy with other anti-cancer agents by drug combination screening in which piperlongumine exhibits broad-spectrum synergistic effects with a large proportion of the tested anti-cancer agents, especially with PI3K/Akt/mTOR pathway inhibitors.

Full text of DOI:10.1039/c9cc00917e

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Activity-based protein profiling reveals GSTO1
as the covalent target of piperlongumine and
a promising target for combination therapy
for cancer†
Cite this:DOI: 10.1039/c9cc00917e
Received 31st January 2019,
Accepted 20th March 2019
Li Li,a Yue Zhao,ab Ran Cao,ac Lin Li,a Gaihong Cai,a Jiaojiao Li,a Xiangbing Qi,a
DOI: 10.1039/c9cc00917e
a
She Chena and Zhiyuan Zhang
*
rsc.li/chemcomm
Through systematic target identification for piperlongumine, a
cancer-selective killing molecule, we identified GSTO1 as its major
covalent target for cancer cell death induction. We also reveal that
GSTO1 inhibition is a promising combination strategy with other
anti-cancer agents by drug combination screening in which
piperlongumine exhibits broad-spectrum synergistic effects with a
large proportion of the tested anti-cancer agents, especially with
PI3K/Akt/mTOR pathway inhibitors.
Given that cancer is the most severe current public health
problem, it is always urgent to discover new molecular targets
and develop effective anti-cancer agents.1 Piperlongumine
(PL-1, Fig. 1a), an alkaloid natural product from Piper longum,
is reported to kill a large variety of cancer cells while remaining
nontoxic to normal cells.2 The cancer cell death induction effect
of PL-1 was demonstrated in in vivo xenograft models of
numerous cancer cell types, indicating that PL-1 is a potential
anti-cancer lead compound. Given its effects on multiple tumor
types, PL-1 must function by inhibiting a cellular target common
to many cancers; this is very promising for anti-cancer drug
development.2,3 PL-1 was shown to interact with glutathione
Fig. 1 PL-1 functions in an irreversible mode. (a) Structure of PL-1–4 and
their cellular activities on NCI-H1975 cells. (b) Cytotoxic activity of PL-1 in
wash/no-wash assays. Under the wash-off condition, NCI-H1975 cells
were incubated with PL-1 for 3 h and then washed with buffer. Cell viability
was measured 24 h after PL-1 administration. (c) The reactivity of PL-1 and
PL-2 with GSH and NAC. NAC/GSH (200 mM) was added to PL-1/PL-2
(10 mM) in medium (physiological conditions) and incubated for 3 h. The
concentration of PL-1/PL-2 was detected by LC-MS/MS before and after
the reaction.
Click-reaction-assisted activity-based protein profiling (ABPP)
S-transferase pi 1 (GSTP1) and carbonyl reductase 1 (CBR1).2a has been developed as a powerful approach to help identify
However, PL-1 has only a modest effect on GSTP1 inhibition, the cellular targets of bioactive molecules.4 Typically, the probe
and the overexpression of GSTP1 and CBR1 showed no rescue molecule is designed to have a terminal alkyne inserted into the
effect on PL-1 induced cell death.2a These reported phenomena bioactive parent molecule that facilitates Cu(I)-catalyzed click
suggest that the selective cancer killing effect of PL-1 might reactions with azido-linked affinity tags. Here, we describe the
result from its interaction with cellular targets other than GSTP1 findings from the first systematic target identification of PL-1
and CBR1. Thus, the functional target and precise molecular using click-reaction-assisted ABPP.
mechanism responsible for PL-1’s selective cancer killing
effects still remain unclear.
Consistent with previous reports, PL-1 treatment of a variety
of cancer cell lines induced cell death with an IC50 at the low
micromolar level (Table S1, ESI†).2 Since the olefins of PL-1
have strong electrophilicity, we speculated that PL-1 might
covalently bind its target proteins through reaction with nucleo-
philic residues, most likely cysteine thiols. To test this hypothesis,
we conducted wash/no-wash experiments and observed that the
IC50 of PL-1 remained the same under both conditions (Fig. 1b),
a National Institute of Biological Sciences (NIBS), Beijing, 7 Science Park Rd.,
ZGC Life Science Park, Beijing, P. R. China. E-mail: zhangzhiyuan@nibs.ac.cn
b College of Biological Sciences, China Agricultural University, Beijing 100193,
China
c School of Life Sciences, Peking University, No. 5 Yiheyuan Road, Haidian District,
Beijing, P. R. China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc00917e indicating that PL-1 functions irreversibly. Ablation of the C2–C3
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olefin in compounds PL-2 and PL-3 resulted in a significant loss of
cytotoxic activity, while ablation of the C7–C8 olefin in compound
PL-4 only resulted in a decreased cytotoxic activity (Fig. 1a and
Table S1, ESI†), similar to a previous report.2b These results suggest
that the electrophilicity of the C2–C3 olefin in PL-1 is essential
for its cytotoxic activity. Similarly, incubating PL-1 with either
glutathione (GSH) or N-acetyl-L-cysteine (NAC) caused a significant
decrease in the PL-1 concentration and resulted in the formation
of Michael addition products under physiological conditions
(Fig. 1c and Fig. S1, ESI†), while PL-2 remained inert to both
GSH and NAC (Fig. 1c). This further confirmed that PL-1 acts via
covalent interactions with its targets at its C2–C3 site.
Although numerous studies have explored the targets of
PL-1 in order to explain the mechanism through which it
selectively kills cancer cells, few have considered its covalent
binding character.2,3c,e,5 PL-1’s reported binding targets GSTP1
and CBR1 were actually identified in affinity purification
experiments using solid-phase matrices conjugated with PL-1
as the bait.2a However, this method precludes the identification of
candidate targets that covalently bind PL-1, which could possibly
explain why PL-1’s binding to GSTP1 and CBR1 apparently only
made a limited contribution to its cytotoxic activity.2a
In order to design an active alkynylated analogue of PL-1
as the probe molecule for click-reaction-assisted ABPP, we
conducted the structure–activity relationship (SAR) study of
PL-1 to find a suitable site for incorporation of an alkyne group.
The SAR study on the phenyl ring position of the PL structure
indicates that this position is able to tolerate structural diversity to
some extent (Table S2, ESI†). We incorporated a terminal alkyne at
the 4-position of the benzyl ring and removed the –OCH3 groups
at the 3- and 5-positions, thereby obtaining PL-5 (Fig. 2a) as the
probe molecule. PL-5 also functions irreversibly (Fig S2, ESI†) and
retains low micromolar activity for cancer cell death induction
(Fig. 2a and Table S3, ESI†).
Fig. 2 Identification of GSTO1 as the covalent binding target of PL-1.
(a) Structure of the ABPP probe PL-5 and the cytotoxic activity of PL-5
on NCI-H1975 cells. (b) Western blotting results of ABPP-enriched
components using PL-5 (50 mM) as the probe and PL-1 (50 mM) as the
competitor molecule on NCI-H1975 cells. (c) Western blot showing that
the known GSTO1 inhibitor KT45 (20 mM) out-competed PL-5 (50 mM)
for binding with GSTO1 in an ABPP experiment with NCI-H1975 cells.
(d) Molecular docking simulation of PL-1 within the catalytic pocket of
GSTO1.
We next performed click-reaction-assisted ABPP with PL-5 as
the probe and PL-1 as the competitor molecule on NCI-H1975 tyrosine residues at this position.7c Two series of GSTO1-
cells (Fig. S3, ESI†). To identify PL candidate targets that were inhibitor compounds have been discovered to date, and both
enriched in the PL-5 labeled samples and were competed out achieve GSTO1 inhibition through binding to the active Cys32
by PL-1, we filtered the protein MS results (43-fold enrichment residue.8 We found here that KT45—one of the reported GSTO1
of the probe-labeled samples versus the out-competed samples; inhibitors—showed micromolar activity in cell death induction
four independent experiments). Glutathione S-transferase in all of the cancer cell lines that we tested (Fig. S4 and Table S5,
omega-1 (GSTO1) was the top-ranking hit in all four experi- ESI†).8a KT45 could also out-compete the binding of PL-5 with
ments and was thus identified as the most likely cellular target GSTO1 in a follow-up ABPP experiment (Fig. 2c), suggesting that
of PL-1/PL-5 (Table S4, ESI†). We also observed an obvious PL-5 PL-1 and PL-5 might share the same binding site as KT45.
mediated enrichment of GSTO1 in western blots and confirmed
that this enrichment could be efficiently out-competed by PL-1 pyridinone modification was present on the C32 site of GSTO1
(Fig. 2b). in the PL-5 pulldown samples (Fig. S5, ESI†). Labeling experi-
Our MS/MS analysis showed that a 5,6-dihydro-2(1H)-
GSTO1 is a member of the omega class of glutathione ments using recombinant GSTO1 with either PL-1 or PL-5 also
S-transferases (GST); these enzymes are responsible for cellular yielded the aforementioned pyridinone modification at the C32
xenobiotic detoxification based on glutathione conjugation.6 site (Fig. S6, ESI†). These results further supported our previous
Recently, GSTO1 was reported to play a role in chemotherapeutic speculation that PL-1 covalently binds its target through C2–C3
resistance and was associated with enhanced aggressiveness of olefin. We next performed a molecular docking simulation
cancer cells, together making GSTO1 an attractive target for study using the crystal structure of GSTO1 (PBD: 4YQV).8b
anticancer drug development.7 GSTO1 is atypical among GSTs PL-1 fits snugly in the catalytic pocket of GSTO1 (Fig. 2d): the
because it harbors a cysteine residue (Cys32 in human) in its carbonyl group at the C1 site of PL-1 forms a hydrogen bond
catalytic center; canonical GSTs have characteristic serine or with Tyr229; the –SH group of Cys32 is 3.8 Å from the C3 atom
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of the C2–C3 olefin in PL-1, and ready for nucleophilic attack; death induction caused by these molecules (Fig. S9a, ESI†),
the Ile131 can form an H–p interaction with the phenyl ring suggesting that the observed increase in ROS is not the direct
of PL-1; and the 3,4,5-tri-OCH3 substituted phenyl ring moiety cause of cell death. This is in accordance with our result that
dives into the pocket, which still has space for structural pretreatment with NAC failed to rescue cells from death
extension, fully consistent with our SAR results.
induced by GSTO1 knockdown (Fig. S9b, ESI†).
Compounds PL-1 and PL-5 but not PL-2 or PL-3 displayed
The overexpression of GSTO1 has been found in various types
inhibition of GSTO1 enzyme activity in cell lysates and did not of cancer, including melanomas, lymphomas, and colorectal
affect the cellular level of the GSTO1 protein (Fig. 3a, b and cancer, among others.8b There have also been reports that GSTO1
Fig. S7, ESI†). We next evaluated how GSTO1 knockdown affects inhibition may sensitize cancer cells to cell death induced by the
the viability of cancer cell lines that are sensitive to PL-1. popular chemotherapy agent cisplatin.7a,b Consistently, several
Treatment of NCI-H1975, U2OS, HeLa, and A549 cells with studies have noted that PL-1 can enhance the effects of other anti-
GSTO1 siRNA significantly decreased the viability of each cell cancer agents (e.g., cisplatin, gemcitabine, etc.).3b,9 Since drug
type (Fig. 3c), indicating the necessity of GSTO1 for maintaining combinations are being applied more and more widely in cancer
the survival of these tested cancer cell lines. Consistently, the therapy regimens—especially to overcome drug-resistance and to
overexpression of GSTO1 reduced the cell death induction effects reduce side-effects—we were very interested in investigating the
of PL-1 (Fig. 3d). Thus, we can conclude that the cancer cell death potential for conceptualizing GSTO1 as a potentially sensitive
induction effect of PL-1 mainly occurs through its binding and target for combination cancer therapies. Thus, an anti-cancer
inhibition of GSTO1 and that decreased levels of GSTO1 are library (Table S6, ESI†) composed of 540 compounds that
deleterious to cancer cell viability.
comprised both chemotherapeutic agents and targeted small
Both GSTO1 knockdown and inhibition of GSTO1 by PL-1 molecule inhibitors were screened for their combination effect
caused increased accumulation of cellular reactive oxygen with PL-1 using Jurkat, NCI-H1975, and HCT116 cells.
species (ROS) (Fig. S8, ESI†). However, addition of ROS scavenger
In order to investigate PL-1’s combination effects with
NAC after PL-1 or KT45 treatment could not attenuate the cell compounds that showed anti-cancer activities, we applied
Chou–Talalay analysis to calculate a drug combination index
(CI) value.10 Strikingly, PL-1 exhibited synergism with a large
proportion (average of B44%) of the active compounds for
each of the three cell lines (Table S7, ESI†). For the NCI-H1975
cells, PL-1 demonstrated synergism for cell death induction
with 52.3% of the active compounds, and most of the combi-
nation displayed clear (CI o 0.7) or even strong (CI o 0.3)
synergistic effects (Tables S7 and S8, ESI†). Similar results were
obtained with the Jurkat and HCT-116 cell lines (42% and 38%,
Tables S7, S9 and S10, ESI†). This broad-spectrum synergistic
effect of PL-1 with other anti-cancer compounds highlights that
GSTO1 inhibition can be viewed as a promising new approach
for combination therapies to treat multiple types of cancers.
Most of the compounds identified that could synergize with
PL-1 (100% in NCI-H1975 cells and HCT-116 cells and 98% in
Jurkat cells) targeted small molecule anti-cancer agents rather
than chemotherapeutic agents (Tables S8–S10, ESI†). Hit
compounds that target the PI3K/Akt/mTOR pathway appeared
most frequently for each of the three cell lines (27% in NCI-
H1975 cells, 19% in Jurkat cells, and 20% in HCT-116 cells)
(Fig. 4a and Tables S8–S10, ESI†). We selected the PI3K inhibitor
pictilisib as an example compound to evaluate the synergism in
detail.11 PL-1 significantly sensitized NCI-H1975 and Jurkat
Fig. 3 Cancer cell death is induced via PL-1’s inhibition of GSTO1.
(a) Effects of PL-1/2/3/5 on GSTO1 catalysed 4-nitrophenacyl glutathione
cells to pictilisib: in the absence of PL-1, pictilisib had an
reduction activity in cell lysate (NCI-H1975 cell). DMSO and KT45 were
IC50 value of 0.16 mM for NCI-H1975 cells, whereas the addition
of PL-1 reduced its IC50 value to 0.015 mM, a 10-fold increase in
applied as negative and positive controls, respectively. (b) Western blotting
of GSTO1 after compound incubation of NCI-H1975 cells for
3 h.
its activity (Fig. 4b). Similarly, experiments with Jurkat cells
showed that combination of pictilisib with PL-1 resulted in an
18-fold increase in activity vs. pictilisib alone (Fig. S10a, ESI†).
Moreover, the sensitizing effect of PL-1 was also apparent on
Akt phosphorylation monitored with western blotting: although
PL-1 itself did not inhibit phosphorylation of Akt and mTOR
(Fig. S11, ESI†), the presence of PL-1 increased the extent of
(c) Effects of GSTO1 knockdown with two different GSTO1 siRNAs (10 nM)
on survival of NCI-H1975, HeLa, U2OS and A549 cells (left to right). Top:
Western blot of GSTO1 after GSTO1 siRNA transfection. Bottom: Cell
survival rate measured after GSTO1 siRNA transfection. (d) Effects of
GSTO1 overexpression on U2OS cells. Left: Western blot of GSTO1 after
transfection. Right: Cell survival rate measured after PL-1 treatment.
(control: transfected with empty plasmid.) *: P value o 0.05; **: P value o
0.01; ***: P value o 0.001.
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GSTO1 alone as well as in combination with other anti-cancer
agents as a promising strategy for cancer treatment.
This work was supported by the National Major Scientific
and Technological Special Project (2013ZX0950910) from the
Chinese Ministry of Science and Technology.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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In conclusion, using the click-reaction-assisted ABPP method,
our study established that PL-1’s covalent binding and inhibition
of GSTO1 can explain its strong cytotoxic effect against multiple
cancer types. We also revealed the promising prospect of GSTO1
inhibition in combination therapies for cancer treatment by
showing that PL-1 exhibits quite broad-spectrum synergistic
effects with many anti-cancer drugs, especially with PI3K/Akt/
mTOR pathway inhibitors. Therefore, as GSTO1 may be a uni-
versal target for many different types of cancers, researchers can
now focus on exploring the potential application of inhibiting
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