Induction of Apoptosis in Cancer Cells by Glutathione Transferase Inhibitor Mediated Hydrophobic Tagging Molecules

Article abstract of DOI:10.1021/acsmedchemlett.0c00627

The abnormally high expression of glutathione transferases is closely associated with cancer incidence and drug resistance. By introducing a hydrophobic moiety to the inhibitor structure, we organized a series of degraders of glutathione transferases and

Full text of DOI:10.1021/acsmedchemlett.0c00627

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Letter
Induction of Apoptosis in Cancer Cells by Glutathione Transferase
Inhibitor Mediated Hydrophobic Tagging Molecules
̈
Xiaona Li, Zirui Lu, Cong Wang, Kebin Li, Fengrong Xu, Ping Xu,* and Yan Niu*
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ABSTRACT: The abnormally high expression of glutathione transferases is closely associated with cancer incidence and drug
resistance. By introducing a hydrophobic moiety to the inhibitor structure, we organized a series of degraders of glutathione
transferases and demonstrated them potently inducing apoptosis in cancer cells, presenting their pharmacological potential in cancer
therapy.
KEYWORDS: glutathione transferase pi, protein degradation, hydrophobic tagging
he glutathione transferases (GSTs) are a group of typical
phase II detoxification enzymes found mostly in the
characterized by high GSTP expression which can be linked
with cancer incidence and drug resistance.8 It is anticipated
that GST, especially the pi class, can be developed as a
biomarker for early cancer detection or diagnosis as well as for
targeted preventive and therapeutic interventions.9 Indeed,
inhibition of GSTP expression through antisense cDNA was
able to increase the cancer cell sensitivity to adriamycin,
cisplatin, melphalan, and etoposide.10 Meanwhile, a significant
number of GST inhibitors has been synthesized to modulate
drug resistance as sensitizers to chemotherapy due to the
predominant overexpression of GSTs in tumor cells.11,12
In recent years, proteolysis-targeting chimera (PROTAC)
has emerged as a useful tool for the chemical knockdown of a
protein of interest (POI). It employs E3 ligase ligands, fused
via a flexible chemical linker to a targeting element for the POI,
to elicit ectopic ubiquitination, resulting in proteasomal
protein degradation.13 Alternatively, replacement of the E3
ligase ligand with a strong hydrophobic molecule like
T
cytosol. These enzymes catalyze conjugations of glutathione
with a wide variety of exogenous and endogenous chemicals
with electrophilic functional groups, forming water-soluble
GSH conjugates that are readily transported out of the cell.
Thus, GSTs play a pivotal role in cellular protection from
environmental and oxidative stress, as well as in cancer cell
resistance to drugs.1 Up to now, seven classes of the
mammalian cytosolic GSTs are recognized, namely, alpha,
mu, pi, theta, sigma, zeta, and omega.2 Many of them also
catalyze isomerase, glutathione peroxidase, or thiol transferase
reactions in the synthesis of leukotrienes, prostaglandins, and
steroid hormones as well as in the degradation of tyrosine and
inactivation or reduction of oxidative stress byproducts. In
addition to their enzymatic activities, several GST family
members also exhibit nonenzymatic functions in the regulation
of cell signaling pathways implicated in stress responses, cell
survival, and apoptosis.1−3 Specifically, GST pi (GSTP) has
been known as an endogenous inhibitor of c-Jun N-terminal
kinase (JNK)4 and tumor necrosis factor receptor associated
factor 2 (TRAF2).5 GSTA1 has been shown to physically
interact with JNK,6 and GSTM1 has inhibitory interaction
directly with the apoptosis signal-regulated kinase 1 (ASK1).7
Of these enzymes, GSTP is of particular interest with regard to
cancer because many tumors and cancer cell lines are
Received: November 27, 2020
Accepted: April 29, 2021
Published: May 3, 2021
https://doi.org/10.1021/acsmedchemlett.0c00627
© 2021 American Chemical Society
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adamantyl, which is known as hydrophobic tagging (HyT), has
been demonstrated as an efficient approach to downregulate
the target protein in many cases.14,15 Considering the
promising pharmacological utility of GSTs, especially GSTP,
regarding tumor genesis and chemoresistance, we wonder
whether chemical downregulation of GSTs could generate a
better therapeutic effect compared with functional inhibition.
In 2012, Hedstrom et al. reported that a kind of chimera
molecule connecting ethacrynic acid (EA), a covalent GST
inhibitor, to Boc3Arg by a (CH2)6 linker (Figure 1), was
Figure 1. Reaction of EA or EA-Boc3Arg with GST.
Figure 2. Structure and synthesis of EA-based HyTs. Reagents and
conditions: (i) 1-adamantanamine hydrochloride, EDCI, HOBT,
NMM, DMF, rt, overnight (92%); (ii) N-tert-butoxycarbonyl-1,6-
hexanediamine (N-tert-butoxy carbonyl-1,2-ethylenediamine/N-tert-
butoxycarbonyl-1,4-butanediamine), EDCI, HOBT, NMM, DMF, rt,
overnight (73−93%); (iii) TFA, DCM, rt; (iv) 1-adamantaneacetic
acid, EDCI, HOBT, NMM, DMF, rt, overnight (69−87%, over 2
steps); (v) ethylene glycol (diethylene/triethylene/tetraethylene
glycol), Et3N, 180 °C, 18 h; (vi) ethacrynic acid, EDCI, DMAP,
DMF, rt, overnight (57−72%).
capable to degrade cytosolic GST up to 80% at 80 μM.16 The
mechanism was later demonstrated through direct localization
of GSTP to 20S proteasome that subsequently executed
degradation.17
In this study, we synthesized a series of HyT molecules that
connect EA with the adamantyl moiety through either multiple
ethylene or diethoxyl chains (Figure 2), aiming at increasing
the hydrophobicity of the GSTP surface and thus triggering the
cell’s protein quality control machinery and ultimately
proteasomal degradation. Synthesis of these compounds was
achieved from commercially available EA in 2 or 3 steps. For
compounds with carbon linker ADC0EA−ADC4EA, EA
reacted with different N-Boc protected diamines to form
amides (3a−3c). Then, the −NH2 groups were released by
removal of protecting groups to react with 1-adamantaneacetic
acid to afford corresponding amides (5a−5c). For compounds
ADE1EA−ADE4EA, 1-bromoadamantane was converted to
ether with different polyethylene glycols with the terminal
−OH, finally reacting with EA to give the targeted compounds
8a−8d (Figure 2).
To ensure that attaching of the adamantyl moiety does not
interfere with EA binding to GSTP, we measured the enzyme
inhibitory activity of these bivalent compounds against GSTP
using 1-chloro-2,4-dinitrobenzene (CDNB) assay in which the
inhibition of GSTP catalyzed a conjugation reaction between
GSH and CDNB, measured by the decrease in absorbance at
340 nm specific to the conjugate.18 Results showed that except
for ADC0EA, which suffered a loss of GSTP inhibition
potency, all other compounds were able to inhibit GSTP
enzymatic activity, especially at high concentration of 100 μM
(Figure 3A). Exposure of Hela (human cervical carcinoma)
cells in which GSTP is expressed in high degree to either 40 or
80 μM of the −CH2− linked compounds for 3 h induced
obvious degradation of GSTP (Figure 3B). However, this was
not the case for diethoxy linked compounds (Figure 3C). The
most potent compound was ADC4EA, which generated over
80% clearance of GSTP protein at 40 μM. Consistent with our
expectation, this compound was observed to dose-dependently
downregulate the GSTP protein level at concentrations higher
than 20 μM (Figure 3D and S1). In contrast, the synthetic
intermediate 3b which carries the C4 linker but no adamantyl
tag exhibited no degradation at all tested concentrations
(Figure 3E). This result emphasized the importance of the
adamantyl tag for degradation. Because the intracellular
protein homeostasis is maintained by a balance of both
protein synthesis and degradation, and to reduce the impact of
GSTP expression on protein level, we further pretreated the
cells with cycloheximide (CHX) to inhibit protein synthesis
before ADC4EA treatment. Indeed, ADC4EA dose-depend-
ently downregulated the GSTP protein level (Figures 3F and
S2), suggesting this downregulation was a result of improved
degradation instead of decreased expression.
We also compared the potencies of ADC4EA and 3b in
GSTP degradation in two more cell lines, namely A549 lung
adenocarcinoma and HT1080 fibrosarcoma cell lines, in which
the GSTP are expressed in high or medium levels.19 Likewise,
ADC4EA was found to degrade GSTP in these two cell lines,
while 3b was not measured at high concentrations (Figure 4B)
To further validate that this degradation was processed
through the ubiquitin-proteasome pathway (UPP), we used
bortezomib, a 20S proteasome inhibitor, to cotreat the cells,
and as expected, the degradation induced by ADC4EA was
significantly reversed. In addition, the degradation can also be
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Figure 4. (A) The GSTP protein levels in A549 cells after treatment
with ADC4EA or 3b for 3 h. (B) The GSTP protein levels in HT1080
cells after treatment with ADC4EA or 3b for 3 h.
Figure 5. (A) The GSTP levels in Hela cells after treatment of
ADC4EA or the combination of ADC4EA with indicated agents for 3
h. (B) Quantitation of panel A by ImageJ and Origin 9.0 software.
The protein levels were measured via Western blot assay with specific
antibodies using total cell lysates. Data are representative of three
independent experiments.
Figure 3. (A) Inhibitory activities of the HyTs against GSTP. The
values are an average of three experiments in parallel. GSTP protein
levels in Hela cells after (B) ADC0EA to ADC4EA and (C) ADE0EA
to ADE4EA treatment for 3 h. Dose−response of GSTP protein levels
in Hela cells treated with (D) ADC4EA or (E) 3b for 3 h. (F) Dose−
response of GSTP protein levels in Hela cells pretreated with CHX
followed ADC4EA treatment for 3 h. The protein levels were
measured in Western blot assay with specific antibodies using total
cell lysates. Data are representative of three independent experiments.
Through activation by cytosol MAPKKs,20 the released JNK is
going to induce chain events, starting from the phosphor-
ylation of c-Jun, resulting in alteration in cell cycle, DNArepair,
and apoptosis.21 Clearly, we have observed increased
phosphorylation of JNK and c-Jun in ADC4EA treated cells,
whereas no obvious alteration was observed in the EA treated
cells (Figure 6A and S3). Interestingly, the total JNK levels
seemed also downregulated by a certain degree in the 20, 40,
or 80 μM treated groups (Figures 6A and B and S4), indicating
the degradation of the entire GSTP−JNK complex induced by
ADC4EA. Considering that reduction of oxidative stress
byproducts is one of the major biofunctions of GST family
members, the GSTP degradation is supposed to result in an
accumulation of the reactive oxygen species (ROS) in cells. We
thus measured the ROS level using DCFH-DA ROS florescent
probe.22 As shown in Figure 6C, there was a significant ROS
accumulation in the ADC4EA (20 μM) treated cells, which was
reversed by PYR-41, which is an inhibitor of ubiquitin-
activating enzyme E1, suggesting the degradation relies on the
ubiquitination of the target protein (Figure 5). Interestingly,
when the cells were cotreated with bafilomycin A1, a specific
inhibitor of vacuolar type H (+)-ATPase that prevents
autophagy at late stage by inhibiting fusion between
autophagosomes and lysosomes, the ADC4EA induced GSTP
degradation was also greatly reversed. This suggested that in
addition to UPP, ADC4EA can also induce GSTP protein
degradation through autophagy pathway.
As an endogenous inhibitor of JNK, GSTP degradation is
speculated to release JNK from the GSTP−JNK complex.
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Figure 7. ADC4EA dose-dependently induced apoptosis in Hela cells.
(A) The percentage of cells in different apoptotic states was measured
by flow cytometric analysis using Annexin V-FITC and PI double-
staining. (B) Quantitation of panel A by Origin 9.0.
triggering apoptotic cell death. Therefore, HyT-based GSTP
degraders might have pharmacological potentials not limited to
sensitizing chemotherapy but also as a single therapeutic agent.
In this study, EA was utilized as a ligand to bind with GSTP.
However, it is known that EA also reacts with other GST
isoenzymes and shows a preference toward GSTP and GSTA1.
The observed degradation is also supposed to happen on other
GST isoenzymes, for example, GSTM (Figure S5). The
induced apoptosis should be the total effect resulting from
degradation of all potential targets, although only GSTP was
emphasized in this study. Because there are also some selective
GSTP inhibitors available, for example the GSH analogue
TLK199, it will be interesting and applicable to develop an
HyT molecule using a selective GSTP inhibitor, by which a
clearer contribution of GSTP to the apoptosis can be observed.
Unlike the specific E3 ligase-based PROTACs, the structure of
the hydrophobic moiety of HyT-based degraders can be very
diverse and simple, facilitating the fast design and synthesis of
the degrader. As reported by Mignani et al.23 that the poor
antiproliferative capacity of EA could be modified by
introduction of relevant lateral chain leading to the activation
of the apoptotic cascade, although the degradation of the GSTs
was not measured in their study. Indeed, we have observed that
addition of a simple hexyl chain to EA had generated a
compound potent enough to induce GSTP degradation at 80
μM (Figure S6). This study also showed that the hydro-
Figure 6. ADC4EA promoted phosphorylation of JNK and c-Jun and
dose-dependently induced increase in ROS level. (A) Hela cells were
treated with ADC4EA for 3 h, followed by Western blot assay with
specific antibodies using total cell lysates. Data are representative of
three independent experiments. (B) Quantitation of total JNK of
panel A by ImageJ and Origin 9.0 software. (C) Hela cells were
treated with the indicated concentrations of ADC4EA before the ROS
level was quantitatively detected as a measure of fluorescence at Ex/
Em: 488/525 with a ultrahigh-resolution laser confocal microscope
(Nikon, Japan).
comparable to the positive control (50 μg/mL of Rosup),
while almost no ROS can be detected in the EA group even at
a concentration as high as 80 μM.
Considering JNK phosphorylation initiates multiple pro-
apoptotic signaling and accumulation of ROS is a strong
stimulus of cellular apoptosis, we further measured the
apoptotic population in the treated cells using flow cytometry.
Dose-dependently, ADC4EA induced apoptosis in cells after
treatment for 3 h (Figure 7A). Treatment with 80 μM of
ADC4EA has generated 61% apoptotic population. In contrast,
only a marginal portion (∼15%) of apoptotic cells was
observed in the EA treated cells (Figure 7B). The above
observations indicated that compared with functional inhib-
ition of GSTP, the HyT induced GSTP degradation should be
more lethal to cancer cells due to its strong potency in
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phobicity of the linker between EA and AD played an
important role in the degradation effect. The hydrophobic
carbon chain was more preferential, and the degradation
potency could be modified by shifting the length of the linker.
In conclusion, by linking an adamantyl moiety to a GST
inhibitor, we developed a series of HyT molecules that can
potently degrade GSTP in cancer cells. Upon GST
degradation, significant activation of JNK and accumulation
of ROS was observed, which finally led to apoptotic cell death.
The degradation was found through both proteasomal and
autophagic pathways that updated the current knowledge
about HyT technique. The work also provided preliminary
structure−activity relationship regarding the HyT linker to
facilitate the design of more degraders with pharmacological
potential.
ACKNOWLEDGMENTS
■
This work was generously supported by the National Natural
Science Foundation of China (Grants 81872731 and
21977006). The authors also appreciate Dr. Paola Ripani
from the Biochemistry Division, University of Konstanz for
advices on GST subtypes.
ABBREVIATIONS
■
GST, glutathione transferase; JNK, c-Jun N-terminal kinase;
TRAF2, tumor necrosis factor receptor associated factor 2;
ASK1, apoptosis signal-regulated kinase 1; PROTAC,
proteolysis-targeting chimera; POI, protein of interest; HyT,
hydrophobic tagging; EA, ethacrynic acid; CDNB, 1-chloro-
2,4-dinitrobenzene; CHX, cycloheximide; UPP, ubiquitin-
proteasome pathway; ROS, reactive oxygen species; EDCI,
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlor-
ide; HOBT, 1-hydroxybenzotriazole; NMM, N-methyl mor-
pholine; DMF, N,N-dimethylformamide; TFA, trifluoroacetic
acid; DCM, dichloromethane; Et3N, triethylamine
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* Supporting Information
The Supporting Information is available free of charge at
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REFERENCES
■
Typical experimental procedures; supporting figures
(S1−S5); synthesis and characterization of target
compounds with NMR and mass spectra (PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
Yan Niu − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China; orcid.org/0000-
0001-9193-8659; Email: yanniu@bjmu.edu.cn
Ping Xu − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China; orcid.org/0000-
0003-2455-6647; Email: pingxu@bjmu.edu.cn
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Authors
Xiaona Li − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China; orcid.org/0000-
0002-7041-2604
Zirui Lu − Department of Medicinal Chemistry, School of
̈
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China; orcid.org/0000-
0003-1448-431X
Cong Wang − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China
Kebin Li − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China; orcid.org/0000-
0002-2120-3662
Fengrong Xu − Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Peking University Health Science
Center, Beijing 100191, P.R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00627
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Notes
The authors declare no competing financial interest.
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