Rational design of platinum(IV) compounds to overcome glutathione-S- transferase mediated drug resistance

Article abstract of DOI:10.1021/ja0432618

A rationally designed Pt(IV) anticancer compound is described, employing the novel concept of tethering an inhibitor of glutathione-S-transferase, an enzyme associated with Pt-based drug-resistance, to cisplatin. Its enzyme inhibition activity, investigat

Full text of DOI:10.1021/ja0432618

Published on Web 01/13/2005
Rational Design of Platinum(IV) Compounds to Overcome
Glutathione-S-Transferase Mediated Drug Resistance
Wee Han Ang,† Isam Khalaila,† Claire S. Allardyce,† Lucienne Juillerat-Jeanneret,‡ and
Paul J. Dyson*,†
Institut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique Fe´de´rale de Lausanne, EPFL-BCH,
CH 1015 Lausanne, Switzerland, and UniVersity Institute of Pathology, Centre Hospitalier UniVersitaire Vaudois,
CH 1011 Lausanne, Switzerland
Received November 9, 2004; E-mail: paul.dyson@epfl.ch
Chart 1
Despite the success of cisplatin and its analogues as anticancer
drugs used in 70% of all cancer treatments, drug resistance remains
one of the most serious and challenging problems to overcome.
Much progress had been made to understand the mechanisms
involved in drug resistance, which are multifactorial processes.1
One important enzyme responsible for drug resistance in some
cancers, is glutathione-S-transferase (GST).2 Cytosolic GST en-
zymes constitute the main cellular defense against xenobiotics, and
they are known to catalyze the conjugation of glutathione (GS-H)
with cisplatin in vitro,3 the first step in the mercapturic acid pathway
that leads to elimination of toxic compounds. Studies also found
GST enzymes, specifically GST-π isozymes, to be overexpressed
in cisplatin-resistant cell lines,4 and the inhibition of these enzymes
has led to the reversal of drug resistance.5
A broad range of GST inhibitors, with varying degrees of
isozyme specificity, are known, and some of them have been
systemically tested in combination with a range of alkylating agents
against multiple drug resistant (MDR) cancers as an adjuvant.6 In
particular, ethacrynic acid (EA), a diuretic in clinical use, has been
Pt(NH3)2Cl2(OH)2 using EA anhydride was also unsuccessful. The
extensively studied and found to effectively inhibit all GST
ability of 1 to inhibit GST activity was studied in vitro using the
isozymes, but to different extents.7 Clinical trials involving EA in
established 1-chloro-2,4-dinitrobenzene (CDNB)-glutathione (GSH)
combination with chlorambucil and thiotepa against a range of
spectrophotometric assay (see Supporting Information). Briefly,
cancers were of some success.8
A549 lung carcinoma cells were exposed to cisplatin, EA, or 1 for
Satraplatin is a promising Pt(IV) anticancer drug,9 currently
a short duration, sufficient for drug uptake, but insufficient for
undergoing Phase III clinical trials for combination treatment in
postexposure modification or cell death, then disrupted to extract
patients with hormone-refractory prostate cancer. It functions as a
their cytosolic contents. The GST activity of the extracts were
prodrug, releasing its cytotoxic Pt(II) payload, after losing its axial
determined and compared to untreated cells. Interestingly, it was
acetato-ligands by reduction in vivo. Satraplatin was even found
found that the GST activity of the A549 cells exposed to 1 decreased
to be suitable for oral administration as it is stable enough to survive
to 22.6% of the control levels, while those exposed to EA and
the harsh conditions in the gastrointestinal environment. Based on
cisplatin decreased to 78.5% and 63.6%, respectively, providing
the studies summarized above, we decided to tether EA to platinum
the first indication that 1 could be a potent GST inhibitor as well
to give a satraplatin-like compound, 1, capable of targeting GST
as an anticancer drug. This could be due in part to higher drug
enzymes in human cancer cells. On uptake, the compound should
uptake in cells treated with 1, which showed 10-fold higher Pt levels
be reduced in vivo releasing the EA moiety (inhibiting the GST
compared to those treated with cisplatin (see Supporting Informa-
enzyme) as well as a cytotoxic Pt(II) center, thus reversing cisplatin-
tion), presumably as a result of higher lipophilicity conferred by
associated drug resistance.
the large organic ligands.12 Based on drug uptake at low temper-
The synthesis of 1 was achieved via the acylation of cis,cis,trans-
ature, 1 appears to enter the cells via passive diffusion.
Pt(NH3)2Cl2(OH)2 with an excess of the EA chloride without the
To eliminate peripheral effects such as cellular drug uptake or
addition of an amine base. This method represents a deviation from
interference from other cytosolic entities, the compounds were tested
a reported procedure in which pyridine is needed to quench the
directly against specific GST isozymes. The inhibition of GSTP1-1
free HCl in the acylation process,10 which failed to give 1. It has
and GSTA1-1 (GST-R isozymes are also associated with drug
been reported that in the absence of an adequate HCl-acceptor the
resistance)13 by the test compounds were studied using the CDNB-
formation of PtIV(amine)2Cl4-x(O2CR)x (x ) 0-2) mixtures takes
GSH assay (see Supporting Information). The results showed that
place.11 However, by controlling and optimizing the reaction
1 is a potent inhibitor, more than EA itself, and capable of reducing
conditions, the target compound can be readily isolated in mod-
the activity of both isozymes to less than 10% of the original
erate yield (see Supporting Information). Acylation of cis,cis,trans-
activity, even at low compound concentration. To our knowledge,
this represents the first example of a Pt(IV) compound tailor-made
to inhibit the activity of relevant drug resistant enzymes. The results
† Ecole Polytechnique Fe´de´rale de Lausanne.
‡ Centre Hospitalier Universitaire Vaudois.
9
1382
J. AM. CHEM. SOC. 2005, 127, 1382-1383
10.1021/ja0432618 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Comparison of Cytotoxicities of Cisplatin and
Ethacraplatin (1) on Selected Cancer Cell Lines
a
IC50
(
µM)
MCF7
24 h 72 h
T47D
24 h
HT29
24 h
A549
24 h 72 h
test compound
72 h
72 h
cisplatin
>80
36.02 >80
58.82 >80
16.82 >80
31.43
ethacraplatin (1) 31.85 32.15 31.56 34.88 32.63 12.96 78.59 32.09
a IC50, drug concentration that inhibits cell growth by 50%. Only the
results for 24 h and 72 h exposure are displayed (see Supporting Information
for other details). Each value is the mean of three independent experiments.
using MTT assays over periods of 24, 48, and 72 h. The growth of
T47D cells decreased at lower concentrations of 1 compared to
cisplatin, and growth inhibition in the other cells was accelerated
on exposure to 1 (Table 1). After 24 h of exposure, growth inhi-
bition was observed in all cells exposed to 1, but not in cells exposed
to cisplatin, suggesting a role for GST in this faster effect of 1. As
GST has been involved in several cellular pathways regulating
growth, besides its known involvement in resistance to chemo-
therapeutics agents,16 further experiments will be necessary to
understand the cell mechanisms involved in the accelerated effect
of 1.
In conclusion, a novel fast-acting cytotoxic Pt(IV) compound
with the capacity to inhibit GST activity has been prepared,
representing a strategy of utilizing the Pt(IV) carboxylate framework
to build customized compounds capable of delivering multiple
modes of pharmacological effects.
Figure 1. Comparison of deconvoluted spectra (ESI-QT) of GSTA1-1
(bottom), GSTA1-1 treated with 10-fold excess of 1 (top), and GSTA1-1
treated with 10-fold excess of EA (middle).
Acknowledgment. We thank the Roche Research Foundation
and Swiss Cancer League for financial support.
also suggest that cisplatin is not capable of inhibiting GSTP1-1,
via active site occupancy or otherwise, which, in vivo, could allow
it to be rapidly conjugated to GSH and deactivated.
Supporting Information Available: Experimental procedures and
spectroscopic data for 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
The interactions between 1 and the GST isozymes were probed
using mass spectrometry. The ESI mass spectrum (deconvoluted
to reveal the parent mass) of GSTA1-1 is shown in Figure 1
(bottom) revealing a parent mass of ca. m/z 25 504. Incubation of
GSTA1-1 with a 10-fold excess of EA leads to a mass spectrum
containing the intact enzyme and an additional peak at m/z 25 807
corresponding to the formation of the enzyme-EA adduct (center).
No adducts were observed when GSTA1-1 was incubated with
cisplatin, but with a 10-fold excess of 1, both the enzyme-EA and
enzyme-1 adducts are observed. The spectrum suggest a GST-
induced cleavage of the Pt-EA carboxylate bond, although these
results give no indication of the mechanism of carboxylate bond
cleavage which may not involve the same catalytic functional
groups as GSH conjugation. This is plausible since GSTP1-1 and
GSTA1-1 have been reported to catalyze the hydrolysis of thiol
esters of EA, albeit at a much slower rate compared to the
conjugation to GSH.14 Furthermore, the crystal structure of GST-
EA complexes show EA bound nonproductively in the active site,
with the carboxylate group directed outward to the solvent region.15
A similar mode of binding may occur with 1, possibly weakening
the carboxylate bond between the Pt(IV) center and EA due to
electronic and steric effects of EA binding. A similar observation
was made when GSTP1-1 was incubated with EA or 1, but with
the formation of multiple adducts. Similarly, no significant interac-
tions with cisplatin were observed.
References
(1) Fuertes, M. A.; Alonso, C.; Perez, J. M. Chem. ReV. 2003, 103, 645-
662.
(2) Niitsu, Y.; Takahashi, Y.; Ban, N.; Takayama, T.; Saito, T.; Katahira, T.;
Umetsu, Y.; Nakajima, T.; Ohi, M.; Kuga, T.; Sakamaki, S.; Matsunaga,
T.; Hirayama, Y.; Kuroda, H.; Homma, H.; Kato, J.; Kogawa, K. Chem.-
Biol. Interact. 1998, 112, 325-332.
(3) Goto, S.; Iida, T.; Cho, S.; Oka, M.; Kohno, S.; Kondo, T. Free Radical
Res. 1999, 31, 549-558.
(4) Ferrandina, G.; Scambia, G.; Damia, G.; Tagliabue, G.; Fagotti, A.; Panici,
P. B.; Mangioni, C.; Mancuso, S.; DIncalci, M. Ann. Oncol. 1997, 8, 343-
350.
(5) Nakajima, T.; Takayama, T.; Miyanishi, K.; Nobuoka, A.; Hayashi, T.;
Abe, T.; Kato, J.; Sakon, K.; Naniwa, Y.; Tanabe, H.; Niitsu, Y. J.
Pharmacol. Exp. Ther. 2003, 306, 861-869.
(6) (a) Schultz, M.; Dutta, S.; Tew, K. D. AdV. Drug DeliVery ReV. 1997, 26,
91-104. (b) Morgan, A. S.; Ciaccio, P. J.; Tew, K. D.; Kauvar, L. N.
Cancer Chemother. Pharmacol. 1996, 37, 363-370.
(7) Takamatsu, Y.; Inaba, T. Toxicol. Lett. 1992, 62, 241-245.
(8) Odwyer, P. J.; Lacreta, F.; Nash, S.; Tinsley, P. W.; Schilder, R.; Clapper,
M. L.; Tew, K. D.; Panting, L.; Litwin, S.; Comis, R. L.; Ozols, R. F.
Cancer Res. 1991, 51, 6059-6065.
(9) Wong, E.; Giandomenico, C. M. Chem. ReV. 1999, 99, 2451-2466.
(10) Galanski, M.; Keppler, B. Inorg. Chem. 1996, 35, 1709-1711.
(11) Giandomenico, C. M.; Abrams, M. J.; Murrer, B. A.; Vollano, J. F.;
Rheinheimer, M. I.; Wyer, S. B.; Bossard, G. E.; Higgins, J. D. Inorg.
Chem. 1995, 34, 1015-1021.
(12) Hall, M. D.; Hambley, T. W. Coord. Chem. ReV. 2002, 232, 49-67.
(13) Tew, K. D. Cancer Res. 1994, 54, 4313-4320.
(14) Dietze, E.; Grillo, M.; Kalhorn, T.; Nieslanik, B.; Jochheim, C.; Atkins,
W. Biochemistry 1998, 37, 14948-14957.
(15) Oakley, A. J.; Rossjohn, J.; Lo Bello, M.; Caccuri, A. M.; Federici, G.;
Parker, M. W. Biochemistry 1997, 36, 576-585.
The growth inhibition of 1 against a range of established cancer
cell lines, including the cisplatin-resistant breast MCF7 and T47D,
lung A549, and colon HT29 human carcinoma cells, was studied
(16) Townsend, D.; Tew, K. Oncogene 2003, 22, 7369-7375.
JA0432618
9
J. AM. CHEM. SOC. VOL. 127, NO. 5, 2005 1383