An inhibitor of glutathione S-transferase omega 1 that selectively targets apoptotic cells

Article abstract of DOI:10.1002/anie.201203730

On (cell) suicide watch: The increased permeability of apoptotic cells was used to identify a peptide-based, covalent inhibitor (NJP2) for glutathione S-transferase omega (GSTO1) that is highly selective for apoptotic cells, with no inhibition of healthy

Full text of DOI:10.1002/anie.201203730

Angewandte
Chemie
DOI: 10.1002/anie.201203730
Peptide Inhibitor
An Inhibitor of Glutathione S-Transferase Omega 1 that Selectively
Targets Apoptotic Cells**
Nicholas J. Pace, Daniel R. Pimental, and Eranthie Weerapana*
The dysregulation of apoptosis plays a critical role in cancer
progression.[1] Cytotoxic anticancer agents function by induc-
ing apoptosis, and chemotherapeutic resistance is tightly
coupled with defective progression of apoptotic cascades.[2] To
identify proteins implicated in maintaining or accelerating
apoptosis, it would be advantageous to develop apoptotic cell-
selective inhibitors with no effect on healthy cells. Such
context-dependent inhibitors are valuable tools to deconvo-
lute protein activities implicated in chemotherapeutic resist-
ance and accelerate apoptosis within a specific cell popula-
tion. Furthermore, a selective covalent modifier of proteins in
apoptotic cells can serve as a tool for imaging cell death.
Toward this end, covalent probes for caspases have been
shown to be selective and versatile agents for imaging
apoptosis in vivo.[3]
To identify apoptotic-cell-selective inhibitors we sought to
exploit the loss of plasma membrane integrity that accom-
panies apoptosis.[4] This phenomenon has been exploited in
the development of apoptosis-selective dyes to image cell
death. For example, the green fluorescent YO-PROR-1 dye
preferentially accumulates in apoptotic cells.[5] Similarly,
a peptide-based organoarsenical compound was shown to
internalize into apoptotic cells at the stage at which plasma-
membrane integrity is compromised.[6] This organoarsenical
compound was comprised of a tri-peptide backbone, suggest-
ing that small peptidic structures could act as a vehicle for
selective delivery of inhibitors into apoptotic cells. To explore
this hypothesis, we sought to investigate the protein-labeling
properties of an electrophilic tri/tetrapeptide library in
healthy and apoptotic cells. Specifically, we conjugated the
peptides to a cysteine-reactive electrophile to identify apop-
totic-cell-selective inhibitors of cysteine-mediated protein
activities.
proteases, oxidoreductases, and metabolic enzymes.[7] These
reactive cysteines can be targeted by small molecules
containing electrophiles such as haloacetamides, Michael
acceptors, and sulfonate esters.[8] A peptide-based library of
chloroacetamides was shown to demonstrate intriguing pro-
teome-labeling patterns, but was not evaluated in whole cells
(in situ).[9] We expand on this previous study by exploring the
protein-labeling properties of cysteine-reactive peptides
in situ, in both healthy and apoptotic cells, with the goal of
inhibiting specific cysteine-mediated protein activities within
an apoptotic cell.
We synthesized a library of cysteine-reactive compounds
comprised of acrylamide or sulfonate ester electrophiles,
a variable peptide region, and an alkyne tag in the form of
a propargyl glycine as a site for click chemistry[10], which
allows for target visualization and enrichment (Figure 1). The
peptide region exploited the inherent structural diversity of
commercially available amino acids. These peptides were
synthesized on a solid-support using standard Fmoc-based
peptide-coupling techniques, and the electrophiles were
subsequently added while the peptide was still on the resin,
using a hydroxypropanoic acid linker to incorporate the
sulfonate ester (see Supporting Information, Scheme S1). Ten
of these peptides were evaluated for apoptosis-selective
labeling events in HeLa cells; cells were incubated with
either dimethyl sulfoxide (DMSO) as a control or stauro-
sporine (STS), a broad-spectrum protein kinase inhibitor, to
induce apoptosis.[11] These control and apoptotic cells were
subsequently treated with the peptide library by adding the
compounds directly to the media of the cultured cells. Protein
labeling by each peptide was analyzed by in-gel fluorescence
after lysing the cells and performing click chemistry to
incorporate a rhodamine-azide (Rh-N3) dye (Supporting
Information, Figure S1).[12]
We were interested in peptides that demonstrated differ-
ential labeling of a single protein between control and
apoptotic cells. One peptide in this initial ten-member library,
NJP2 (Figure 2a), satisfied these criteria by labeling a 28 kD
band in apoptotic cells, with no detectable protein labeling in
the control cells (Figure 2b). To further characterize this
unique labeling profile, we performed a time-course analysis
of STS treatment while monitoring the extent of apoptosis by
DNA fragmentation (Figure 2c, top panel). The intensity of
protein labeling by NJP2 increased proportionally with the
progression of apoptosis (Figure 2c, bottom panel). Further-
more, we treated Jurkat cells with STS to show that this
labeling event was not specific to HeLa cells (Supporting
Information, Figure S2). Finally, cells treated with the DNA-
topoisomerase inhibitor, camptothecin (CPT), confirmed that
Highly reactive cysteines serve functional roles in catalysis
and protein regulation in a diverse array of proteins, such as
[*] N. J. Pace, D. R. Pimental, Prof. E. Weerapana
Department of Chemistry, Merkert Chemistry Center, Boston
College
2609 Beacon Street, Chestnut Hill, MA 02467 (USA)
E-mail: eranthie@bc.edu
[**] Eranthie Weerapana is a Damon Runyon–Rachleff Innovator
supported (in part) by the Damon Runyon Cancer Research
Foundation (DRR-18-12). We are also grateful for financial support
from the Smith Family Foundation and Boston College. We thank
Prof. Jianmin Gao, David Alex Shannon, and other members of the
Weerapana Lab for comments and critical reading of the manu-
script.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201203730.
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this labeling event occurs under other chemically
induced models of apoptosis (Supporting Infor-
mation, Figure S3).
After demonstrating the apoptotic-cell selec-
tivity of NJP2, we identified the 28 kD protein
targeted by NJP2. NJP2-labeled lysates under-
went click chemistry to incorporate biotin-azide,
followed by purification on streptavidin beads,
on-bead trypsin digestion, and LC/LC–MS/MS
analysis.[13] Proteins identified in the NJP2-
treated lysates were compared to DMSO-treated
lysates by mass spectrometry. This analysis
enabled us to identify glutathione S-transferase
omega 1 (GSTO1) as the protein target of NJP2,
because high spectral counts (142 and 155) were
observed in duplicate NJP2-labeled samples,
with no spectral counts in the DMSO-treated
samples (Supporting Information, Table S1).
Figure 1. The sulfonate ester (NJP1–5) and acrylamide (NJP6–10) electrophile-bearing
peptides screened for apoptosis-selective protein labeling in HeLa cells.
Spectral counts refer to the total number of fragmentation
spectra found to correspond to peptides from GSTO1. The
molecular weight of GSTO1 (27566 Da) also coincides with
the migration of the apoptosis-selective band observed in our
fluorescent gel analyses.
GSTs catalyze the conjugation of glutathione (GSH) to
a variety of exogenous and endogenous electrophiles as
a cellular defense against chemical carcinogens, therapeutic
drugs, and oxidative stress.[14] Of the seven cytosolic GST
classes, GSTO1 is unusual in that it contains a cysteine residue
in the active site (C32), in place of the canonical tyrosine or
serine residues found in the other mammalian GSTs.[15]
GSTO1 is overexpressed in highly aggressive human cancers
and is implicated in chemotherapeutic resistance.[16]
To confirm GSTO1 as the protein target of NJP2, we over-
expressed both the wild-type and catalytic-cysteine mutant
(C32A) GSTO1 in HEK293T cells by transient transfection
(Figure 2d). NJP2 treatment followed by in-gel fluorescence
analysis demonstrated extensive labeling of the wild-type
protein by NJP2. No labeling was detected for the C32A
mutant (Figure 2d), which demonstrated that covalent modi-
fication of GSTO1 by NJP2 occurs at the active site cysteine.
Next, we investigated the mechanism of the apoptotic cell
selectivity of NJP2. To confirm our hypothesis that the
selectivity is due to increased permeability of the apoptotic
cell membrane, we had to eliminate the possibility of an
increase in GSTO1 abundance or activity during apoptosis. A
previously reported proteomic study into proteolysis events
occurring during apoptosis demonstrated no change in
GSTO1 levels.[17] To confirm this result and eliminate the
possibility of post-translational activation of GSTO1 during
apoptosis, we compared protein labeling in control and
apoptotic cells using a non-specific sulfonate ester activity-
based probe (PS-alkyne). This probe is known to be cell-
membrane permeable and to label GSTO1, amongst other
targets.[18] This study confirmed that GSTO1 abundance and
activity remain unchanged during apoptosis (Supporting
Information, Figure S4), suggesting that the labeling of
GSTO1 in apoptotic cells is due to selective internalization
of NJP2, resulting from the compromised integrity of the cell
membrane.
Figure 2. a) Chemical formula of NJP2; b) In-gel fluorescence of con-
trol and apoptotic cells (4 mm STS for 4 h) labeled with NJP2 (50 mm
for 1 h). c) DNA-fragmentation assay monitoring the extent of apopto-
sis (top panel) and in-gel fluorescence from NJP2 labeling (bottom
panel) at various time points after inducing apoptosis. d) Mock-
transfected, GSTO1-wild-type, and GSTO1-C32A mutant-expressing
cells labeled with NJP2 and analyzed by in-gel fluorescence (top panel)
and western blot (bottom panel; anti-myc antibody). Overexpressed
GSTO1 runs at a higher molecular weight compared to endogenous
GSTO1 (*) owing to the presence of a C-terminal myc/His tag and
spacer.
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To demonstrate selective internalization of NJP2 in
apoptotic cells, we synthesized a fluorescent analog (NJP13)
by adding a rhodamine using click chemistry (Figure 3a).
Control and apoptotic cells were treated with NJP13 and
visualized by fluorescence microscopy. STS-treated cells,
when exposed to NJP13, displayed a significant fluorescence
increase, above background fluorescence levels. The intensity
of the fluorescence increased with the extent of apoptosis and
coincided with the morphological changes in cellular struc-
ture that accompany apoptosis (Figure 3b). In contrast, no
fluorescence was observed in the NJP13-treated control cells
(Figure 3b). This dramatic increase in cellular uptake upon
induction of apoptosis confirms our hypothesis that the
increased cell-permeability during apoptosis can be exploited
to selectively deliver peptide-based molecules into apoptotic
cells.
Figure 4. Monitoring the extent of GSTO1 inhibition by NJP2. Control
and STS-treated cells were dosed with increasing concentrations of
NJP2 (see bottom for concentrations) and the resulting lysates were
a) treated with Rh-N3 or b) treated with PS-Rh and analyzed by in-gel
fluorescence. c) Quantitation of GSTO1 activity as measured by com-
petitive ABPP upon treatment of control and STS-treated cells with
NJP2 (data shown is a representative plot from two repeat experi-
ments). %=Percent of GSTO1 activity remaining.
to GSTO1 activity, and thereby a loss in PS-Rh labeling
signifies inhibition of GSTO1 activity.[19] Control and apop-
totic cells were treated with increasing concentrations of
NJP2, ranging from 1–60 mm. The resulting lysates were either
subjected to click chemistry with Rh-N3 or treated with PS-Rh
and visualized by in-gel fluorescence (Figure 4a,b, respec-
tively). The Rh-N3 gels illustrate the dose-dependent increase
in GSTO1 labeling in apoptotic cells (Figure 4a, top panel),
with minimal labeling in control cells (bottom panel). Label-
ing of GSTO1 appears to be saturated at 40 mm NJP2, and
even at the highest concentration assayed there was negligible
labeling of proteins other than GSTO1 (Supporting Informa-
tion, Figure S5). In the competitive ABPP experiment, the
decrease in the PS-Rh labeling of GSTO1 coincides with an
increase in the Rh-N3 signal (Supporting Information, Fig-
ure S6). Complete inhibition of GSTO1 in apoptotic cells is
achieved at approximately 40 mm (Figure 4b, top panel) with
no decrease in GSTO1 activity of control cells (Figure 4b,
bottom panel). Integration of the band intensity at each
concentration quantifies the residual GSTO1 activity and
demonstrates the striking selectivity of NJP2 towards
GSTO1-inhibition in apoptotic cells (Figure 4c). These data
demonstrate that NJP2 reacts specifically with GSTO1 only
within apoptotic cells.
Figure 3. Increased membrane permeability of apoptotic cells.
a) Chemical formula of NJP13. b) Fluorescence microscopy images of
HeLa cells incubated with DMSO (control) or STS (1 mm) for 30 min,
1 h, or 2 h, followed by NJP13 treatment (1 mm for 1 h).
To investigate the potency and selectivity of GSTO1
inhibition by NJP2 in control and apoptotic cells, we
performed a dose-dependent labeling experiment and a com-
petitive activity-based protein-profiling (ABPP) experiment.
The competitive ABPP experiment used a fluorescently
tagged phenyl sulfonate (PS-Rh) probe to determine residual
GSTO1 activity after treatment with NJP2.[19] The degree of
PS-Rh labeling of GSTO1 has been shown to be proportional
In summary, we report a peptide-based GSTO1 inhibitor
that is selectively internalized in apoptotic cells and functions
through specific covalent modification of the active-site
cysteine of this enzyme. Owing to the role of GSTO1 in
drug resistance, it is highly desirable to find selective
inhibitors for this enzyme. Toward this goal, several inhibitors
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itors that are equipotent for GSTO1 in both healthy and
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GSTO1. This inhibitor, NJP2, is highly selective for targeting
the enzyme in apoptotic cells. Despite the low affinity of NJP2
toward GSTO1 in apoptotic cells (IC50 ꢀ 13 mm; Supporting
Information, Figure S7), the high specificity it displays for
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Received: May 15, 2012
Published online: && &&, &&&&
Keywords: apoptosis · glutathione S-transferase omega ·
.
inhibitors · peptides · protein modifications
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Peptide Inhibitor
N. J. Pace, D. R. Pimental,
E. Weerapana*
&&&& — &&&&
An Inhibitor of Glutathione S-Transferase
Omega 1 that Selectively Targets
Apoptotic Cells
On (cell) suicide watch: The increased
permeability of apoptotic cells was used
to identify a peptide-based, covalent
inhibitor (NJP2) for glutathione S-trans-
ferase omega (GSTO1) that is highly
selective for apoptotic cells, with no
inhibition of healthy cells (see scheme).
This apoptosis-specific peptide could also
be used for imaging programmed cell
death.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!