Overcoming the drug resistance in breast cancer cells by rational design of efficient glutathione s-transferase inhibitors

Article abstract of DOI:10.1021/ol902298s

"Chemical Equation Presented" A new type of competitive human GST inhibitors has been developed via the bioisostere and structure activity profile strategies; we report their discovery, preparation, inhibitory activity, and synergetic effect in combination with chemotherapy drugs against breast cancer cells.

Full text of DOI:10.1021/ol902298s

ORGANIC
LETTERS
2010
Vol. 12, No. 1
20-23
Overcoming the Drug Resistance in
Breast Cancer Cells by Rational Design
of Efficient Glutathione S-Transferase
Inhibitors
Wen-Shan Li,*,† Wing See Lam,† Kung-Cheng Liu,†,‡ Chie-Hong Wang,†
Hui Chuan Chang,† Ya Ching Jen,† Yu-Ting Hsu,† Sachin S. Shivatare,†,§ and
Shu-Chuan Jao⊥
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan, Department of Chemistry,
National Taiwan Normal UniVersity, Taipei 116, Taiwan, Chemical Biology and
Molecular Biophysics Program, Taiwan International Graduate Program, Academia
Sinica, Taipei 115, Taiwan, and Institute of Biological Chemistry, Academia Sinica,
Taipei 115, Taiwan
wenshan@chem.sinica.edu.tw
Received October 5, 2009
ABSTRACT
A new type of competitive human GST inhibitors has been developed via the bioisostere and structure activity profile strategies; we report
their discovery, preparation, inhibitory activity, and synergetic effect in combination with chemotherapy drugs against breast cancer cells.
Drug resistance plays an important role in the success or
failure of anticancer therapies. The development of drug
resistance is a major disadvantage for the use of chemo-
therapeutic drugs (e.g., cisplatin, thiotepa, chlorambucil,
doxorubicin) in cancer treatments.1 Many approaches have
been tried to elucidate the possible mechanisms involved in
drug resistance. A key proposed mechanism relates to the
participation of mutidrug-resistance-related proteins (MRP)
and P-glycoprotein (P-gp), which effectively remove a wide
range of endogenous electropliles and exogenous compounds
(e.g., anticancer drugs) out of the cytosol to reduce the
intracellular toxicity.2 In addition, glutathione S-transferases
† Institute of Chemistry, Academia Sinica.
(1) For a recent review, see: (a) Hayes, J. D.; Pulford, D. J. Crit. ReV.
Biochem. Mol. Biol. 1995, 30, 445–600. (b) Townsend, D. M.; Tew, K. D.
Oncogene 2003, 22, 7369–7375. (c) Zhao, G.; Wang, X. Curr. Med. Chem.
2006, 13, 1461–1471.
‡ National Taiwan Normal University.
§ Taiwan International Graduate Program, Academia Sinica.
⊥ Institute of Biological Chemistry, Academia Sinica.
10.1021/ol902298s  2010 American Chemical Society
Published on Web 12/04/2009

(GSTs, E.C.2.5.1.18), a family of GSH-dependent enzymes,
contribute importantly to drug resistance by catalyzing adduct
formation between glutathione (GSH) and anticancer drugs
(Figure 1).3 GSTs in mammals were originally divided into
strategies have been utilized to identify GST inhibitors,
including a convenient NMR-based screening,8 dynamic
combinatorial chemistry,9 peptidomimetic GSH-conjugates,10
and Pt(IV) carboxylate framework.3d Although several suc-
cessful inhibitor candidates of GSH analogues have been
developed from these methods, they are typically restricted
to GSH-ethacrynic acid derivatives7c,e or prepared through
lengthy chemical procedures. Furthermore, some of the GSH
analogues display poor permeability across the plasma
membrane, and their clinical applications for inhibiting drug
resistance are relatively limited. Herein we report the first
rapid discovery of GST inhibitors via the bioisostere strategy
and discover the capability of lithocholic acid as one of
elements in GSH-type inhibitor design to enhance cell
permeability. In addition, we demonstrated that the lead
compound 3 shows synergetic effect with chemotherapy
drugs against breast cancer cells, and our approaches should
pave the way for the design of effective GST inhibitors.
Figure 1. Possible GST-mediated activation of anticancer drug
resistance in cancer cells.
In the initial design of GSH-based analogues, we chose
γGlu-Ser-X as the framework (Scheme 1), which is capable
eight different classes on the basis of their biofunctional
properties and sequence identities.4 In particular, it has been
shown that different GST isoenzymes such as GST P1-1,
GST A2, and GST M1 are overexpressed in many cancer
cell lines including breast cancer.5 Therefore, GST inhibition
has been recognized as an important strategy for suppression
of GST-mediated anticancer drug resistance and regulation
of cell signaling processes.
Scheme 1. Compound 1 Composed of Lithocholic Acid (LA)
Moiety Is the Lead Selected by GST Assay
To date, two types of inhibitors of GSTs, non-GSH
compounds6 and GSH analogues,7 have been reported, and
some of them have been demonstrated to enhance the
cytostatic effect of numerous anticancer drugs. Distinctive
(2) (a) Broxterman, H. J.; Giaccone, G.; Lankelma, J. Curr. Opin. Oncol.
1995, 7, 532–540. (b) Pe´rez-Toma´s, R. Curr. Med. Chem. 2006, 13, 1859–
1876.
(3) (a) Tew, K. D. Cancer Res. 1994, 54, 4313–4320. (b) Frankmoelle,
W. P.; Medina, J. C.; Shan, B.; Narbut, M. R.; Beckmann, H. Drug Metab.
Dispos. 2000, 28, 951–958. (c) Depeille, P.; Cuq, P.; Mary, S.; Passagne,
I.; Evrard, A.; Cupissol, D.; Vian, L. Mol. Pharmacol. 2004, 65, 897–905.
(d) Ang, W. H.; Khalaila, I.; Allardyce, C. S.; Juillerat-Jeanneret, L.; Dyson,
P. J. J. Am. Chem. Soc. 2005, 127, 1382–1383. (e) Turella, P.; Filomeni,
G.; Dupuis, M. L.; Ciriolo, M. R.; Molinari, A.; De Maria, F.; Tombesi,
M.; Cianfriglia, M.; Federici, G.; Ricci, G.; Caccuri, A. M. J. Biol. Chem.
2006, 281, 23725–23732.
of searching bioactive component to target GST enzymes.
With the oxygen in place for sulfur atom, the serine residue
should be in a position to mimic the cysteine of GSH with
the absence of ability as a real substrate. The synthesis of
the γGlu-Ser-X series was started with condensation of Boc-
Glu(Ot-Bu)-Ser-OH and X (aromatic, aliphatic, and hetero-
cyclic amines/alcohols), followed by the removal of Boc and
t-Bu group. Individual components from the library were
purified by HPLC and tested as possible inhibitors for GSTA2,
GSTM1, and GSTP1-1, representative of enzymes of corre-
sponding human R, µ, and π classes. Analyses of the screening
results lead to a rapid identification of a new optimal binding
component, lithocholic acid (LA). As is evident from Table 1,
compound 1 (γGlu-Ser-LA) displayed low inhibition ability
(4) Nebert, D. W.; Vasiliou, V. Hum. Genomics 2004, 1, 460–464.
(5) (a) McIlwain, C. C.; Townsend, D. M.; Tew, K. D. Oncogene 2006,
25, 1639–1648. (b) Lo, H. W.; Ali-Osman, F. Curr. Opin. Pharmacol. 2007,
7, 367–374.
(6) (a) Wu, Z.; Minhas, G. S.; Wen, D.; Jiang, H.; Chen, K.; Zimniak,
P.; Zheng, J. J. Med. Chem. 2004, 47, 3282–3294. (b) Ricci, G.; De Maria,
F.; Antonini, G.; Turella, P.; Bullo, A.; Stella, L.; Filomeni, G.; Federici,
G.; Caccuri, A. M. J. Biol. Chem. 2005, 280, 26397–26405. (c) Turella, P.;
Cerella, C.; Filomeni, G.; Bullo, A.; De Maria, F.; Ghibelli, L.; Ciriolo,
M. R.; Cianfriglia, M.; Mattei, M.; Federici, G.; Ricci, G.; Caccuri, A. M.
Cancer Res. 2005, 65, 3751–3761. (d) Ang, W. H.; Parker, L. J.; De Luca,
A.; Juillerat-Jeanneret, L.; Morton, C. J.; Lo Bello, M.; Parker, M. W.;
Dyson, P. J. Angew. Chem., Int. Ed. 2009, 48, 3854–3857.
(7) (a) Lyttle, M. H.; Hocker, M. D.; Hui, H. C.; Caldwell, C. G.; Aaron,
D. T.; Engqvist-Goldstein, A.; Flatgaard, J. E.; Bauer, K. E. J. Med. Chem.
1994, 37, 189–194. (b) Cacciatore, I.; Caccuri, A. M.; Cocco, A.; De Maria,
F.; Di Stefano, A.; Luisi, G.; Pinnen, F.; Ricci, G.; Sozio, P.; Turella, P.
Amino Acids 2005, 29, 255–261. (c) Mahajan, S.; Atkins, W. M. Cell. Mol.
Life Sci. 2005, 62, 1221–1233. (d) Jao, S. C.; Chen, J.; Yang, K.; Li, W. S.
Bioorg. Med. Chem. 2006, 14, 304–318. (e) Burg, D.; Riepsaame, J.; Pont,
C.; Mulder, G.; van de Water, B. Biochem. Pharmacol. 2006, 71, 268–
277.
(8) Lo, W. J.; Chiou, Y. C.; Hsu, Y. T.; Lam, W. S.; Chang, M. Y.;
Jao, S. C.; Li, W. S. Bioconjugate Chem. 2007, 18, 109–120.
(9) Shi, B.; Stevenson, R.; Campopiano, D. J.; Greaney, M. F. J. Am.
Chem. Soc. 2006, 128, 8459–8467.
(10) Burg, D.; Filippov, D. V.; Hermanns, R.; van der Marel, G. A.;
van Boom, J. H.; Mulder, G. J. Bioorg. Med. Chem. 2002, 10, 195–205.
Org. Lett., Vol. 12, No. 1, 2010
21

OH and Boc-Glu(Ot-Bu)-OH to give protected 3 followed
by removal of the remaining Boc and t-Bu groups (Scheme
2, compounds 2, 4, and 5; see Supporting Information).
Table 1. Inhibitory Constants of Glutathione Mimic with LA
Moiety against Different GSTsa
inhibitory activity, IC50 (µM)
compound
GSTA2
GSTM1
GSTP1-1
Scheme 2. Synthesis of SAR Probes for Human GSTs
GSH
440.0 ( 32.0b
>100 (22)e
10.4 ( 1.1
3.6 ( 0.3
120.0 ( 20.0c
15.9 ( 0.6
3.8 ( 0.6
16.3 ( 2.1
(4.6 ( 1.6)f
11.6 ( 1.0
6.1 ( 0.3
37.2 ( 7.4
87.7 ( 2.7
<200 (55)h
>50 (46)i
125.0 ( 6.0d
>100 (0)e
1
2
3
>100 (0)e
1.4 ( 0.2
(0.6 ( 0.1)f
>100 (9)e
4
5
6
7
8
9
>100 (0)e
>300 (0)g
>300 (5)g
188.5 ( 14.8
>200 (27)h
>200 (0)h
<200 (52)h
>200 (20)h
>200 (0)h
>50 (25)i
a Inhibitor concentration at which half-maximal enzyme activity is
obtained (IC50, mean ( SEM, n ) 3). b Value (Km) is from ref 11. c Value
(Km) is from ref 12. d Value (Km) is from ref 13. e The % inhibition, in
parentheses, at 100 µM is expressed as the % inhibition of enzyme activity.
f Values reported are Ki. g The % inhibition, in parentheses, at 300 µM is
expressed as the % inhibition of enzyme activity. h The % inhibition, in
parentheses, at 200 µM is expressed as the % inhibition of enzyme activity.
i The % inhibition, in parentheses, at 50 µM is expressed as the % inhibition
of enzyme activity.
against GSTA2, and no inhibition was detected against
GSTP1-1 at concentrations as high as 100 µM. By contrast,
this molecule is a highly selective inhibitor against GSTM1
with IC50 ) 15.9 ( 0.6 µM, suggesting that compound 1 might
be an acceptable probe in the study of GSTM1-mediated drug-
resistant malignancies. For comparison, Table 1 lists the
substrate Km values of GSH for different GST isoenzymes.
Because of the overexpression of different GSTs in tumor
cells, discovery of the compounds inhibiting multiple GST
isozymes is therefore a high priority. Since compound 1
represents an effective core structure, we used it as the lead
compound for the development of structure activity profile
of GST inhibitors. To enhance the potency, we explored the
serine residue of compound 1 with alterations of a series of
amino acids, i.e., phenylalanine, aspartic acid, alanine, and
methionine (Figure 2). As a representative example, synthesis
Among all these compounds, compound 3 was the most
active compound with an IC50 of 3.6 and 1.4 µM toward
GSTA2 and GSTP1-1, respectively. In the case of GSTM1
inhibition, compounds 2 and 5 exhibit a 3- to 4-fold potency
increase over 3, while 4 exhibits a slight 1.4-fold increase,
suggesting that adding hydrophobicity is important for
promoting affinity. Interestingly, the results reveal that
compound 5 is at least 2.5-fold more potent than 1 to serve
as a selective GSTM1 inhibitor. Moreover, compound 5 was
50-fold more selective and active against GSTM1 (IC50
)
6.1 µM) than against GSTA2 (5% inhibition in the presence
of 300 µM of 5) and GSTP1-1 (0% inhibition in the presence
of 300 µM of 5).
The inhibition characteristics of compound 3 were evalu-
ated using steady-state kinetic analysis in the presence of
different concentrations of GSH. As shown by Lineweaver-
Burk plots (Figure 3), compound 3 inhibits GSTA2 and
GSTM1 in a competitive manner with Ki ) 0.6 ( 0.1 and
4.6 ( 1.6 µM, respectively (Table 1). The type of inhibition
we observed for compound 3 indicates that substrate (GSH)
and inhibitor compete for the same active site (G-site)
simultaneously. Together, the results serve to explain why
LA-based analogues, γGlu-aa (amino acid)-LA, are a new
alternative use for GSH in GST inhibitor design.
To further explore structure-activity relationships (SAR),
four derivatives (6-9) of compound 3 (γGlu-Asp-LA) with
systematic variations were prepared as depicted in Scheme
Figure 2. Lead optimization process for discovery of 3.
of compound 3 was performed by Fmoc solution-phase
peptide synthesis approach on 10 using Fmoc-Asp(Ot-Bu)-
(12) Gustafsson, A.; Pettersson, P. L.; Grehn, L.; Jemth, P.; Mannervik,
B. Biochemistry 2001, 40, 15835–15845.
(13) Lo Bello, M.; Oakley, A. J.; Battistoni, A.; Mazzetti, A. P.;
Nuccetelli, M.; Mazzarese, G.; Rossjohn, J.; Parker, M. W.; Ricci, G.
Biochemistry 1997, 36, 6207–6217.
(11) Johansson, A. S.; Mannervik, B. J. Biol. Chem. 2002, 277, 16648–
16654.
22
Org. Lett., Vol. 12, No. 1, 2010

Figure 3. Lineweaver-Burk inhibitory analysis of (a) GSTA2 and
(b) GSTM1 steady-state kinetics by compound 3. (a) Reciprocal
velocity versus reciprocal GSH concentration with inhibitor 3
(0, 0.5, 1.5, 3.0, 6.0 µM). CDNB concentration was held constant
at 1 mM. (b) Reciprocal velocity versus reciprocal GSH concentra-
tion with inhibitor 3 (0, 5, 10, 20, 40 µM). CDNB concentration
was held constant at 1 mM.
Figure 4. Synergetic effects of compound 3 on cell viability of
human breast cancer cells MCF-7 and MDA-MB231.
line was favorably resistant to cisplatin and, to lesser extent,
to thiotepa, when compared to MDA-MB-231 cell line. For
each cell line, compound 3 was found to have negligible
effect on proliferative inhibition in the range 0-50 µM.
These observations strongly suggest that compound 3
enhances the antitumor efficacy of cisplatin/thiotepa in breast
cancer cells by blocking the pathway of GST-mediated drug
resistance rather than involving a direct antiproliferative/
cytotoxic effect.
We utilized two complementary strategies, bioisostere
design and structure-activity profile, to identify new and
effective GST inhibitors with acceptable cell permeability.
More importantly, the lead compound 3 shows synergetic
effect with chemotherapy drugs against two breast cancer
cell lines through the inactivation of GST isozymes. Exten-
sion of this approach is designed to explore high-affinity GST
inhibitors, which is of great value in pharmacological and
clinical applications.
2. Surprisingly, replacement of the aspartic acid with
glutamic acid 6 (γGlu-Glu-LA), gave a 2- to 52-fold less
potent inhibitor (Table 1) than 3 (γGlu-Asp-LA), indicating
that adding a methylene group can diminish affinity. More-
over, modification and removal of the γGlu-postion of 3
dramatically decreased potency (7-9). This result is in line
with the previous reports that the γglutamyl moiety of GSH
is highly important in GSTs recognition.
Reversal of drug resistance by inhibiting GST isozymes
has been extensively studied for therapeutic use. To further
demonstrate this specific impact of the GST inhibitor on
anticancer drug cytotoxicity, we investigated compound
3-mediated regulation of cytostatic effect in Vitro. Inhibition
of cell viability by cisplatin and thiotepa in the presence of
GST inhibitor 3 (0, 25, and 50 µM) against two breast cancer
cells, MCF-7 and MDA-MB-231, was studied using MTT
assays. A dose-dependent inhibitory effect on cell viability
was observed, and respective IC50 values of cisplatin or
thiotepa were determined after 48 h (Figure 4, see Supporting
InformationTable S1). The maximal enhancement of cis-
platin-induced inhibition of cell viability was observed at
50 µM compound 3, up to 640% (i.e., decrease in IC50 value
by 6.4-fold) against MCF-7 and up to 270% (i.e., decrease
in IC50 value by 2.7-fold) against MDA-MB-231. Next,
viability inhibition of thiotepa was enhanced by compound
3 (25 and 50 µM), up to 170-320% against MCF-7 and up
to 180-270% against MDA-MB-231. Thus, the MCF-7 cell
Acknowledgment. We are grateful to the National Science
Council (NSC 95-2113-M-001-018-MY3) and Academia
Sinica (program project AS-95-TP-AB1) for their financial
support. The authors thank Ping-Yu Lin and Dr. Mei-Chun
Tseng for mass spectroscopic analysis.
Supporting Information Available: Human GST assay,
IC50 values, general experimental procedures, and compound
characterization. This material is available free of charge via
the Internet at http://pubs.acs.org.
OL902298S
Org. Lett., Vol. 12, No. 1, 2010
23