Identification of a glutathione peroxidase inhibitor that reverses resistance to anticancer drugs in human B-cell lymphoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2012.08.091

Cancer cells isolated from two patients with malignant non-Hodgkin B-cell lymphomas that became resistant to chemotherapy during clinical treatment were made ≥fourfold resistant in culture to anticancer drugs, that is cisplatin, etoposide, methotrexate and bortezomib. Because most resistant lines showed significantly increased expression of the anti-oxidative enzyme glutathione peroxidase 1 (GPx1), GPx1 was investigated as a target for inhibitor development. Virtual screening of a library of diverse structures by docking them to the active site of the X-ray crystal structure of bovine GPx1 uncovered compounds that might block the enzyme. An enzyme assay confirmed an acylhydrazone heterocycle (3) with GPx inhibitory activity. Combinations of 3 with the anticancer drugs listed above led to reversal of resistance in the lymphoma cell lines.

Full text of DOI:10.1016/j.bmcl.2012.08.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6712–6715
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a glutathione peroxidase inhibitor that reverses resistance
to anticancer drugs in human B-cell lymphoma cell lines
Riad Schulz a, Thomas Emmrich a, Heidi Lemmerhirt a, Ulrike Leffler a, Kristin Sydow a, Carsten Hirt b,
Thomas Kiefer b, Andreas Link a, Patrick J. Bednarski a,
⇑
a Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Ernst-Moritz-Arndt University Greifswald, 17487 Greifswald, Germany
b Clinic for Haematology and Oncology, Ernst-Moritz-Arndt University Greifswald, 17475 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Cancer cells isolated from two patients with malignant non-Hodgkin B-cell lymphomas that became
resistant to chemotherapy during clinical treatment were made Pfourfold resistant in culture to antican-
cer drugs, that is cisplatin, etoposide, methotrexate and bortezomib. Because most resistant lines showed
significantly increased expression of the anti-oxidative enzyme glutathione peroxidase 1 (GPx1), GPx1
was investigated as a target for inhibitor development. Virtual screening of a library of diverse structures
by docking them to the active site of the X-ray crystal structure of bovine GPx1 uncovered compounds
that might block the enzyme. An enzyme assay confirmed an acylhydrazone heterocycle (3) with GPx
inhibitory activity. Combinations of 3 with the anticancer drugs listed above led to reversal of resistance
in the lymphoma cell lines.
Received 12 July 2012
Revised 23 August 2012
Accepted 25 August 2012
Available online 5 September 2012
Keywords:
Anticancer drug
Inhibitor
Resistance
Glutathione peroxidase
Non-Hodgkins’s lymphoma
Ó 2012 Elsevier Ltd. All rights reserved.
Non-Hodgkin’s lymphomas (NHL) are a heterogeneous group of
lympho-proliferative disorders with varied aggressiveness and
many therapeutic options. In 2010 approximately 65,540 new
cases of non-Hodgkin lymphoma were diagnosed in the United
States1 and 20,210 died of their disease.2 Depending on the type
of NHL, frequency of relapse is variable. In diffuse large cell lym-
phoma, the most common histologic subtype of NHL, more than
30% will ultimately relapse. Of those responding to salvage therapy
including high dose chemotherapy with stem cell transplantation,
only half of the patients were in continuous complete remission at
a median follow-up of two years. In comparison, the two-year
overall survival rate of patients not responding to salvage therapy
is less than 10 percent. Thus, resistance to chemotherapy in NHL
poses a serious clinical problem.
Over the last decades much has been learned about the mecha-
nisms of resistance to chemotherapy in NHL. Among the more fre-
quent causes are: (1) overexpression of the P-glycoprotein drug
transporter,3 (2) elevated expression of the anti-apoptotic protein
bcl-2,4 and (3) mutations in the tumor suppressor p53.5 Evidence
is growing that the redox state of cancer cells can also have pro-
found effects on the signal transduction pathways leading to pro-
grammed cell death.6 In particular, oxidative mechanisms,
stimulated by the intracellular overproduction of reactive oxygen
species (ROS), in particular hydrogen peroxide, can lead to oxida-
tion of signally proteins such tyrosin phosphatases, kinases and
transcription factors.6,7 Key links have been established between
oxidative stress and the expression of drug transporters8 and
p53.9 Methods for stimulating oxidative stress within cancer cells
to overcome drug resistance have been discussed.10
Intracellular H2O2 levels are mainly regulated by three enzymes
that metabolize it to non-toxic oxygen species: (1) catalase, a heme
enzyme compartmentalized in the peroxisome, disproportionates
H2O2 to H2O and O2,11 (2) peroxiredoxins, which utilize thiore-
doxin as a co-substrate to reduce H2O2 to water,12 and (3) glutathi-
one peroxidases (GPx), which utilize glutathione (GSH) to reduce
both H2O2 as well as organic peroxides and are divided into seven
isoforms, five of which are selenocysteine dependent enzymes.13
The distribution of GPx in the body and within the cells depends
on the isoform, with the most prevalent form being GPx1 (cGPx),
a cytosolic enzyme located in most tissues, with particularly high
levels in erythrocytes, liver, kidney, and lung.14
There have long been reports that increased expression of GPx
in some cancer cell lines is correlated with resistance to anticancer
drugs such as doxorubicin,15 paclitaxel,15b cisplatin,15b bleomy-
cin16 and topotecan.17 Indeed, patients with diffuse large B-cell
lymphoma had a poorer prognosis if their tumor cells overexpres-
sed GPx1.18 It appears that overexpression of GPx protects cancer
cells from apoptosis,19 thus implicating GPx as an anti-apoptotic
protein. A key goal of this project was to investigate the changes
of GPx expression in NHL cell lines made resistant to commonly
used anticancer drugs in culture.
⇑
Corresponding author. Tel.: +49 3834 86 4883; fax: +49 3834 86 4802.
E-mail address: bednarsk@uni-greifswald.de (P.J. Bednarski).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.091

R. Schulz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6712–6715
6713
Table 1
Mean IC50 values (lM) ( SD of 3 or more independent experiments) of four anticancer drugs, determined by the MTT assay, in wild-type (WT) and the respective drug resistant
GUMBUS and DOGUM cell lines, without and with co-incubation with 25
l
M 3. Numbers in parenthesis are the resistance factors for the various drugs in the resistant cell lines.
Drug/
GUMBUS
DOGUM
Resistance
WT
WT + 3
Resistant line
Resistant line + 3
WT
WT + 3
Resistant line
Resistant line + 3
MTX
Etop
CDDP
Bort
0.024 0.010## 0.013 0.001 0.534 0.196##,⁄
0.111 0.093⁄ (5) 0.084 0.012###
0.066 0.022
0.34 0.079###,⁄⁄⁄
(4)
0.087 0.030⁄⁄⁄
(1)
(22)
0.042 0.012### 0.031 0.009 0.57 0.29###,⁄⁄⁄
(13)
0.019 0.004⁄⁄⁄
(0.5)
0.075 0.001+++,### 0.034 0.013+++ 0.28 0.058###,⁄ (4) 0.17 0.060⁄ (2)
0.21 0.07###
0.095 0.06 1.93 0.50###,⁄⁄⁄
(9)
0.18 0.12⁄⁄⁄ (0.8) 0.64 0.08##
0.64 0.49
6.77 2.62##,⁄ (11) 2.46 0.93⁄ (4)
0.006 0.001
0.004 0.002 0.035 0.043 (6)
0.005 0.002 (0.8) 0.002 0.001###
0.002 0.001
0.007 0.001###,⁄⁄⁄ 0.002 0.001⁄⁄⁄
(4)
(1)
Significance determined by two-tailed t-test: ⁄) p < 0.05; ⁄⁄,##) p < 0.02; ⁄⁄⁄,###,+++) p < 0.01.
Drug resistance was re-induced into two non-Hodgkin’s Burkitt
lymphoma cell lines, DOGUM and GUMBUS, previously isolated
from patients with clinical chemoresistance.20 STR analyses re-
vealed a unique DNA profile for both lines, hence derivation from
other established cell lines can be ruled out. To regenerate resis-
tance in vitro, four chemotherapeutics used in clinical therapy
and having different mechanisms of action; that is, methotrexate
(MTX), etoposide (Etop), cisplatin (CDDP), and bortezomib (Bort),
were administered to cells in culture over 2–3 months. A microti-
ter-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay was used to measure the cytotoxicity of the
anticancer drugs21, quantified as the IC50 value (50% inhibitory
concentration). Cell lines were considered resistant when the resis-
tance factor (i.e.; the IC50 in the resistant cells divided by the IC50 in
the wild-type [WT] cells) was four or greater. With the exception of
Inhibition of GPx could have therapeutic potential in slowing or
stopping the development of resistance to anticancer drugs. A
number of GPx inhibitors are already known and include thiols
such as mercaptosuccinate22 and coenzyme A,23 gold complexes
gold(I)–thioglucose24 and auranofin,25 the natural product 2-phen-
ylethyl isothiocyanate26, and the radiosensitizer misonidazol.27
These compounds are either unsuitable for use as therapeutics
(mercaptosuccinate, coenzyme A), have high toxicities (auranofin,
misonidazol) or activate other anti-oxidant pathways (2-phenyl-
ethyl isothiocyanate). Thus, we sought new GPx inhibitors that
were more drug-like, had low inherent toxicity and would reverse
acquired resistance to anticancer drugs in human non-Hodgkin’s
lymphoma cells.
For this purpose, the X-ray crystal structure of GPx1 from bo-
vine erythrocytes,28 which is 90% homologous to the human en-
the bortezomib-resistant GUMUS line, the increases in the IC50 val-
ues of the resistant lines relative to the WT lines were statistically
significant. (Table 1)
zyme, was used in a structure-based approach to screen
thousands of structures for potential GPx1 binding behavior. Nor-
mally, GPx1 is a homotetramer but for our calculations just the
homodimer that forms the catalytic active site was used. Further-
more, the oxidation state of the selenocysteine (SeH) was changed
to a selenenic acid (SeOH) since this is the active form of the en-
zyme following oxidation. The library of structures (ca. 150,000)
was taken from the ZINC database29 with lead-like properties
and automatically docked into the active site of bovine erythrocyte
GPx1 by the AutoDock 4 software package.30 The parameters for
SeOH were added to the force field.31 To score the docked struc-
To quantify the expression of GPx1 in the various WT and resis-
tant cell lines, Western blotting was performed and the level of
staining of the GPx1 bands at 23 kDa compared to the control b-ac-
tin bands at 45 kDa were determined. Representative blots are
shown in Figure 1A, where it can be seen that the GPx1 bands of
the resistant DOGUM cells are more intense compared to WT cells.
In the resistant cell lines the expression of GPx1 increased up to
2.5-fold compared to the WT ones (Fig. 1B); these increases were
statistically significant (p < 0.05) except for MTX-resistant GUM-
BUS cell and the Bort-resistant cell lines. Thus, our results confirm
earlier reports that GPx levels generally increase during resistance
to common anti-cancer drugs.13-17
tures the function
D
G of AutoDock was used and the ligand effi-
G of glutathione was set as an upper
ciency derived;32 the
D
threshold to identify potential hits. Another parameter used was
docking stability; that is, only docked conformations were ac-
Figure 1. (A) Representative Western blot of GPx1 in WT, MTX and Etop resistant DOGUM cell lines; (B) Comparison of GPx1 expression in resistant GUMBUS and DOGUM
cells relative to WT cells. Expression level in WT cells set to 100%. Results are averages SD of at least 3 independent experiments. (⁄p < 0.05 compared to WT by two-tailed t-
test).

6714
R. Schulz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6712–6715
50
250
l
l
M). The concentration of 3 was then varied between 50 and
M to determine the IC50-value and a value of 169 39 M,
l
estimated by linear regression analysis, was obtained. The inhibi-
tory effect of 3 was not a result of the inhibition of GR; this was
proven in a specific GR assay33 up to a concentration of 200
lM.
After confirming enzyme inhibition, the theoretical binding
behavior of 3 in the active site of bovine GPx1 was studied in more
detail. Two binding modes for 3 were apparent, both of which were
embedded in a narrow, secondary binding pocket adjacent to the
broad active site of the catalytic tetrad containing the selenenic
acid (shown in Fig. 2A). In the simulation binding mode just one
was preferred. The methyl imidazole was orientated inwards, an-
chored by an H-bond to a serine (SER176) (Fig. 2B). Another H-
bond formed between the hydrazone moiety and the backbone of
a leucine (LEU139). The hydroxyl group of the phenol was slightly
exposed. Not only was this the most frequently found binding
mode with both AutoDock and MOE, but one with the lowest best
binding energy in AutoDock as well. However, this proposed orien-
tation was only the third lowest energy structure in MOE. In the
second binding mode the compound was flipped in the pocked
with the phenol pointing into the pocket. In this case the phenol
formed an H-bond with SER176 and the hydrazone moiety with
a threonine (THR141). The binding energy from AutoDock was
slightly weaker and not found as often as the first mode. This bind-
ing orientation was the best one suggested from MOE. Both bind-
O
O
K2CO3
THF
N
N
Br
N
NH
O
O
1
Figure 2. (A) Modeled bovine GPx1 showing the active site with the best docking
result from AutoDock (orange) and the third-best docking result from MOE (green).
(B) Binding interactions of the best docking result from AutoDock. Ligand exposure
highlighted in blue. H-bonds can form with SER176 and LEU139. The third bested
docked structure from MOE was almost identical.
H2NNH2 /
MeOH
OH
O
Δ
O
N
N
N
N
H
OH
3
O
O
N
N
NH2
N
cepted that were found in more than half of the cases, for example
in at least 5 out of 10 runs. The allowed size of the first cluster was
set to 5. The best ligands in this selection were merged with the top
ligands ranked by ligand efficiency. Once potential hits were found,
the results were re-docked with AutoDock with the number of runs
increased to 100, and the number of generations and population
size of the Lamarckian genetic algorithm (LGA) as well as the prob-
ability of performing local search being increased as well. The
Molecular Operating Environment (MOE) from the Chemical Com-
puting Group was used to confirm the hits found with AutoDock.
Results from MOE were obtained with the docking simulation algo-
rithm based on the AMBER99 force field, which was modified with
the selenenic acid parameters.31 Ligands were placed in the cata-
lytic site by the Triangle Matcher of MOE, and scoring and rescor-
ing were done with London dG. This led to a selection of 24
substances for inhibitor screening (see supplementary data).
An enzyme assay was used to screen compounds for GPx inhib-
itory activity. The assay has been described previously33 but was
adapted to a 96 well microtiter plate format. It is based on the
GPx catalyzed oxidation of GSH to GSSG by H2O2 followed by
reduction of GSSG by glutathione reductase (GR) back to GSH with
consumption of NADPH, monitored at kmax = 340 nm. Triton X-100
(0.1%) was added to all incubations to prevent precipitation of the
test compounds during the assay, which can lead to non-specific
inhibition of enzymes.34 The assay identified two of the 24 com-
pounds having weak but statistically significant GPx inhibitory
activity; one was N-[(E)-(4-hydroxyphenyl)-methyleneamino]-2-
(2-methyl-imid-azol-1-yl)acetamide (3) (15% inhibition at
N
N
H
NH
N
EtOH / Δ
2
OH
Scheme 1. Synthetic route to 3.
Figure 3. Concentration dependent effect of 3 on the growth of DOGUM (circles)
and GUMBUS (squares) cell lines. Results show averages with SD of three
independent determinations relative to untreated controls.

R. Schulz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6712–6715
6715
ing modes differ little in binding energy; the ratio between them
was ca. 3:2.
While 3 was initially obtained from a commercial source, nei-
Acknowledgments
We thank Anne Schüttler and Gudrun Schuster for technical
assistance and the Rector of the University for special funding.
ther the structure nor the synthesis have been reported in the
literature. The compound was prepared by alkylation of
2-methyl-1H-imidazole with ethyl 2-bromoacetate to give ethyl
2-(2-methylimidazol-1-yl)acetate (1), which was subsequently re-
acted with hydrazine to yield 2-(2-methylimidazol-1-yl)acetohyd-
razide (2) followed by condensation with 4-hydroxybenzaldehyde
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.091.
to give 3 in a 20% overall yield (Scheme 1).35 As reported earlier36
,
the conformational behavior of N-acyl hydrazones results in com-
plex NMR spectra; due to this the 1H-NMR as well as the 13C NMR
spectra show a duplication of signals in the case of 3 (see supple-
mentary data). However, only the docking behavior of the trans
isomer to GPx1 was investigated.
The ability of 3 to reverse resistance in GUMBUS and DOGUM
cells that were resistant towards either MTX, Etop, CDDP or Bort
was explored with the MTT assay. A concentration of 25 lM of 3
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Table 1 summarizes the IC50 values for the four anticancer drugs
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The original hypothesis that a GPx1 inhibitor could lead to an
increase in the total oxidative stress within cells was assessed by
a flow cytometric assay based on the intracellular oxidation of
the dye 20,70-dichlorofluorescin-diacetate (DCFH-DA) by ROS to
the fluorescent 20,70-dichlorofluorescein (DCF). However, these re-
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effect may in part be due to the weak GPx inhibitory activity of
3, which was tested at 250 lM. It could also be that local increases
in H2O2 levels as a result of GPx inhibition are dealt with by other
anti-oxidative enzymes such as catalase and peroxiredoxins, lead-
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activation of various anti-oxidative pathways may be a more sen-
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investigations.
Nonetheless, a lead structure has been identified that reverses
resistance of B-cell lymphoma lines to therapeutically important
anticancer drugs. Structure activity relationship studies on both
GPx1 inhibition and reversal of resistance are currently underway,
which should help to clarify the relationship between GPx1 inhib-
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molecular docking methods can be useful tools in identifying
new GPx inhibitors.
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