Molecular modeling, synthesis, and preliminary biological evaluation of glutathione-S-transferase inhibitors as potential therapeutic agents

Article abstract of DOI:10.1021/jm0504609

Changes in the GSH/GST system have been found to correlate with resistance to anticancer alkylating agents, presumably through accelerated detoxification of these drugs since some GSTs have been shown to catalyze the conjugation of GSH to specific antineo

Full text of DOI:10.1021/jm0504609

6084
J. Med. Chem. 2005, 48, 6084-6089
Molecular Modeling, Synthesis, and Preliminary Biological Evaluation of
Glutathione-S-Transferase Inhibitors as Potential Therapeutic Agents
Antonio Procopio,*,† Stefano Alcaro,† Sante Cundari,§ Antonio De Nino,§ Francesco Ortuso,† Paolo Sacchetta,|
Alfonso Pennelli,| and Giovanni Sindona§
Dipartimento di Scienze Farmaco-Biologiche, Universita` della Magna Graecia, Complesso Ninı` Barbieri, 88021 Roccelletta di
Borgia (Cz), Italy, Dipartimento di Chimica, Universita` della Calabria, Ponte Bucci, cubo 12C, 87036 Arcavacata di Rende
(Cs), Italy, R&D Department, Sigma Tau i.f.r. SpA, via Pontina km 30.400, 00040 Pomezia (Rome), Italy, and Dipartimento di
Scienze Biomediche, Universita` “G. d’Annunzio” Chieti Via dei Vestini, 65100 Chieti, Italy
Received May 16, 2005
Changes in the GSH/GST system have been found to correlate with resistance to anticancer
alkylating agents, presumably through accelerated detoxification of these drugs since some
GSTs have been shown to catalyze the conjugation of GSH to specific antineoplastic agents.
GSH-alkyl derivatives were designed by molecular modeling, synthesized, and tested as
inhibitors of human GST-Pi.
Alkylating agents are a class of clinically useful
anticancer drugs whose efficacy lies in altering DNA
replication by causing single- or double-stranded DNA
breaks and/or cross-linking.1 Despite their considerable
clinical success, significant problems remain that limit
the utility of this modality for cancer treatment, notably
the development of drug resistance.1,2 Generally, resis-
tance may be one of two types: intrinsic or acquired.
The latter is conferred by cellular alterations that occur
as a result of drug exposure, providing cells with
selective survival advantages.
In the case of alkylating agents, resistance has
been attributed to multiple factors including impaired
cellular uptake of drug and altered levels of γ-L-
glutamyl-L-cysteinylglycine (glutathione, GSH) and glu-
tathione-S-transferases (GSTs). Glutathione is the most
abundant nonprotein thiol in the cell with concentra-
tions ranging from 30 µM in plasma to 3.0 mM in the
kidney proximal tubules and levels which may reach
10 mM in tumors of various organs.3 The GSTs are a
family of multifunctional isoenzymes that are widely
distributed throughout mammalian systems. At least
eight different types of human GST have been identified
from isozyme families (Alpha, Mu, Pi, Sigma, Theta,
Zeta, Omega, Kappa).
GSTs catalyze the conjugation of a wide variety of
carcinogenic, mutagenic, toxic, and pharmacologically
active electrophiles to the cellular nucleophile GSH,
producing metabolites that are generally less toxic and
more readily excreted.5
Alkylating agents are very often potent electrophiles,
and many reports have characterized these drugs as
substrates for GSTs. Changes in the GSH/GST system
have been found to correlate with resistance to anti-
cancer alkylating agents, presumably through acceler-
ated detoxification of these drugs. Some GSTs have been
shown to catalyze the conjugation of GSH to specific
antineoplastic agents. (e.g. melphalan, chlorambucil,
cyclophosphamide, BCNU, mechloretamine, etc.).6
Overexpression of GST isozymes has been reported
in a number of different human malignancies, including
cancers of the lung,7 colon,7b,8 kidney,7b,9 ovary,8b,9b
esophagus,10 and stomach.7a,10 This phenomenon can be
considered as a typical adaptive cellular response to
protect vital cellular nucleophiles from drug-induced
damage,11 leading to increased survival and enhanced
resistance to chemotherapy. The link between drug
response and GST expression suggests there is potential
for the use of GST inhibitors in modulating the efficacy
of electrophilic cancer drugs. Several glutathione ana-
logues that target this system have been developed and
have been used both experimentally and clinically in
an attempt to improve efficacy (glutathione-S-trans-
ferase inhibitors and prodrugs, glyoxalase I inhibitors,
and S-nitrosoglutathione).
Concomitant examination of the crystal structure of
the GST isozymes and putative GSH binding, together
with the synthesis of glutathione analogues, provided
the basis for the development of intracellular competi-
tors.12 A broad class of both competitive and noncom-
petitive inhibitors of GST already exists (ethacrinic acid,
BSO, GSH analogues, etc.)12c,13
The EA-GSH conjugate was found to be an enzyme
inhibitor, affecting the GST R-catalyzed reaction be-
tween chlorambucil and GSH.14 This supported the
prediction that GST inhibitors interfere with GST-
mediated conjugation of alkylating agents, modulating
the efficacy of these drugs.15
Adang et al.16 synthesized a series of glutathione
analogues containing a sulfydryl group in which the
C-terminal glycine was replaced by different amino
acids. These compounds were then used as cosubstrates
in reactions catalyzed by rat R and µ GSTs with
1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic sub-
strate. Also, the conjugation of aromatic compounds to
GSH gives products which can function as cosubstrates
for GST,13a,17 and GST-π specific inhibitors were also
proposed.13,18 However, therapeutic limitations includ-
ing toxicity, carcinogenicity, or unsuitability as a drug
* To whom correspondence should be addressed. Tel. +390961391131;
Fax: +390961391270. E-mail: procopio@unicz.it.
† Universita` della Magna Graecia.
‡ Universita` della Calabria.
§ Sigma Tau i.f.r. SpA.
| Universita` “G. d’Annunzio” Chieti Via dei Vestini.
10.1021/jm0504609 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/23/2005

Glutathione-S-Transferase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6085
mon moiety (compound 5). Discrepancies in the affinity
were attributed to the different thiol substituents. To
rationalize the effect of chemical modification, the
computational work was carried out using two different
approaches. The first step was dedicated to the evalu-
ation of the GSH common skeleton enzyme recognition.
The interactions of each isolated substituent with the
GST were studied by means of the program GRID v. 20
Figure 1.
23
using four different probes mimicking the nature of
thiol substituent (1-4): C3 for the sp3 carbon atoms,
C1d for the sp2 carbon atoms (i.e. phenyl groups), O1
for the alcoholic oxygen atoms, and O for the sp2 oxygen
atoms. These calculations were performed with no
ligand in the GST active site, using a resolution equal
to 1 Å (directive NPLA ) 1). GRID results were
considered with two purposes: first to verify the overlap
of the original PDB ligand (GSH, compound 5) in its
crystallographic positioning, and second to analyze
areas, close to 5, suggesting other productive interac-
tions. As depicted in Figure 2, where C3 maps were not
displayed due to the high similarity to those obtained
with the C1d probe, the GRID calculation accuracy was
confirmed, identifying the position of several GSH
groups pertinent to each probe. Areas close to compound
5, where new moieties could be located, particularly
suggested modification of the thiol moiety. Actually, the
region around this group was indicated both by hydro-
philic (N1 and O1) and hydrophobic (C3 and C1d)
probes. Moreover, this region proved sterically able to
locate new substituents. The second step considered the
recognition of compounds 1-5 within the GST catalytic
site using the 6GSS original PDB dimer as receptor.
Docking studies started building our compounds into
the 6GSS chain A active site by introducing the ap-
propriate chemical moieties onto the thiol group of 5
located at its crystallographic positioning. For all com-
pounds, both carboxylic groups of the GSH common
skeleton were considered to be in the ionized form. In
compound 4, taking into account physiological pH and
the presence of two ionized groups, the carboxylic
terminus of the substituent chain was considered to be
in its neutral form. For compounds 2, 3 and 4, both the
R and S configurations of the thiol substituent were
modeled and reported, with r and s suffixes, respectively
(i.e. 2r, 2s, 3r, 3s, 4r, and 4s). 5 was additionally
included for validation purposes. All complexes were
energy minimized using 2000 steps of the Polak-Ribiere
Conjugated Gradient algorithm with the AMBER* force
field 24 and the implicit model of solvation GB/SA water
25 as implemented in MacroModel v. 7.2. 26 During these
optimization procedures, our molecules were allowed to
fully relax their internal degree of freedom, but protein
atom coordinates were kept constrained with a constant
force equal to 100 kJ/mol‚Å.
for human use have inspired the design and synthesis
of new inhibitors, and the best results were obtained
by way of GSH analogues.The technique of developing
enzyme inhibitors based on substrates is widely used
in medicinal chemistry via the principle of microscopic
reversibility in the Michaelis-Menten scheme, which
states that, in a system at equilibrium, any molecular
process and the reverse of that process occur, on the
average, at the same rate.13a,e Many inhibitors are
substances that structurally resemble the enzyme sub-
strate, but either do not react or react very slowly in
comparison. A competitive inhibitor acts by reducing the
concentration of free enzyme available for substrate
binding. Since several studies have described alkyl and
aryl epoxides conjugated to GSH in the presence of
GST,19 we designed our original series of analogues by
using an empirically based method and functionalizing
glutathione as S-alkyl derivatives. These can be con-
sidered GSH-conjugated substituted oxiranes (Figure 1).
These molecules were modeled and synthesized, and
their ability to inhibit human GST-π, elevated in many
tumor types,20 was evaluated in vitro.
Molecular Modeling. Molecular modeling studies
were performed to investigate the interaction between
GST and compounds 1-4 (Figure 1).
The identification of the enzyme model to be used in
our simulation was conducted using the 45 models of
human GST crystallographic structures deposited into
the Protein Data Bank (PDB).21 We focused our atten-
tion onto the GST-Pi isoform complexes with GSH or
its analogues founding 17 structures. The comparison
of the 17 PDB models confirmed a high degree of con-
servation in the 3D structures of residues involved in
the catalytic site. The recognition of all ligands was
almost identical, as were intermolecular contributions,
hydrogen bonding, and electrostatic and hydrophobic
contacts.
For our simulations, we decided to adopt the 6GSS
22
model.
This choice was justified by structural com-
parisons among 6GSS, 8GSS, and 19GS that were the
lower resolution GST-Pi GSH complexes. 8GSS was
discarded because it showed the interaction of three
GST monomers complexing GSH. 6GSS was preferred
to 19GS since this last model reported not only GSH
recognition but also other small organic molecules, such
as the bromosulfalein, located close to the binding site
interacting with both GSH and Phe8.
To analyze the differences among the ligands com-
plexed of all models reported in Figure 1S (see Support-
ing Information), the root-mean-square deviation (RMSd)
was computed considering only the atomic coordinates
of the substructure defined by the GSH common scaffold
(Figure 2 in Supporting Information).
To study the interaction of compounds 1-5 with GST,
5000 conformations of the ligand, starting from the
optimized one, were generated into the enzyme catalytic
site for each complex, using the Monte Carlo method
(applied to all thiol substituent rotatable bonds). The
exclusion of the dihedral angles belonging to the GSH
common skeleton from the Monte Carlo search was
implemented after the RMSd analysis computed onto
the PDB model, reported in Figure 2, that revealed a
remarkable rigidity of this substructure.
The PDB analysis indicated a common enzyme inter-
action pathway for all ligands driven by the GSH com-

6086 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Procopio et al.
Figure 2. GRID maps computed onto 6GSS model (A: N1 at -6.5 kcal/mol; B: C1 ) at -2.5 kcal/mol; C: O at -5.0 kcal/mol;
D: O1 at -6.5 kcal/mol). For clarity only residues within 5Å from the GSH (reported in ball and stick notation) have been displayed.
*From chain B.
Table 1. Docking Resultsa,b
good agreement with experimental Ki values (see Table
1), allowing a preliminary validation of our docking
approach. Interestingly, ∆Gs indicated, for chiral sub-
stituted compounds 2-4, weak effects of their thiol side
chain configurations on GST affinity. The docking
structural analysis was extended to the comparison of
the 5 Monte Carlo results versus the crystallographic
model 6GSS by computing the RMSd between theoreti-
cal and crystallographic models.29 Remarkably, we
observed an almost complete reproduction of the ex-
perimental geometry as reported by the RMSd value
equal to 0.147 Å. Such a result, taking into account the
exhaustive exploration of compounds 1-5 GST recogni-
tion, reported by the AND data, and the good agreement
between theoretical and experimental affinity data,
indicated satisfactory quality of our docking approach.
After the validation steps, with the aim to investigate
the different binding modes and GST affinities of
compound 1-5, we analyzed the global minimum energy
configuration generated by docking studies considering
their Boltzmann population. The GSH common skeleton
of 1-5 compounds revealed the same kind of interaction.
Specifically, we observed in all cases at least two
intramolecular hydrogen bonds between the terminal
carboxylic moieties the their R-located amide groups.
Compounds 1, 2r, 2s, and 3r showed a third intramo-
lecular hydrogen bond between the hydroxyl group
positioned onto the thiol substituent and the GSH
glutamyl γ sp2 oxygen atom. All ligands displayed three
intermolecular hydrogen bonds among the GSH Gly
carboxyl terminus and Trp38, Gln51, and Lys44. This
last residue also gave a significant electrostatic contri-
bution to the complex stabilization. Another intermo-
lecular hydrogen bond was observed between the GSH
Cys carbonyl group and Leu52 backbone amide. Such a
residue, through its carbonyl group, was also involved
compound
RB
NCONF
AND
GM%
∆G
1
2r
2s
2rs
3r
3s
3rs
4rq
4s
7
8
8
- -
8
8
- -
7
7
- -
2
1038
633
868
- -
656
1165
- -
3.03
4.91
3.51
- -
4.66
2.85
- -
99.18
76.52
92.59
- -
99.74
90.76
- -
-63.32
-69.23
-68.28
-68.76
-72.71
-72.62
-72.67
-69.71
-68.08
-68.90
-57.52
511
325
- -
8.81
5.27
- -
99.77
82.10
- -
4rs
5
3
2.00
98.89
a RB ) number of rotatable bonds analyzed by Monte Carlo;
NCONF ) total number of configuration; AND ) average number
of duplicates; GM% ) Boltzmann population of the global mini-
mum energy structure; ∆G ) estimated free binding energy in
kcal/mol. b Data of compounds rs were computed as the average
of the r and s.
All generated structures were submitted for energy
multiminimization using the same force field and
solvent environment previously described. In this case,
to take into account the induced fit phenomena, the
positional constant force constrain was applied only to
the GST backbone atoms. A preliminary docking evalu-
ation was carried out considering the total number of
generated structures within 50 kJ/mol above the global
minimum energy (NCONF), their distribution, and the
average number of duplicate structures (AND). In our
experience, an AND value equal or higher than 2
indicates a good exploration of the conformational
space.27 According to the MOLINE method,28 final
geometries were used for estimation of the free energy
of binding ∆G (Table 1).
AND values revealed a sufficiently complete explora-
tion of the conformational space for all compounds.
Moreover, theoretical thermodynamic data revealed a

Glutathione-S-Transferase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6087
Scheme 1
in a second hydrogen bond with the GSH Cys amide.
These important interactions were also reported be-
tween the GSH amine terminus and Gln64. A highly
productive contribution was revealed for the γ glutamyl
carboxyl group, which accepted one hydrogen bond from
Ser65 and a second from Arg13salso involved from an
electrostatic point of viewsand two with the backbone
and terminus amides of Asn66. Finally, the highly
functionalized GSH common skeleton also showed hy-
drophobic contacts between its methylene γ glutamyl
side chain and Pro53. The most populated geometric
analysis followed with the interaction between thiol
substituents and the GST binding site. Compound 1
revealed an intermolecular hydrogen bond and van der
Waals contact, respectively, between the alcoholic and
methylene groups and Arg13. 2r and 2s, due to a
rotation of the thiol bond, demonstrated large binding
mode similarity. In particular, both molecules showed
the same interactions reported for 1 plus a π-π contact
between their aromatic ring and Tyr108. Compounds
3r and 3s reported a different recognition. 3r showed
binding mode interaction similar to that indicated for
compounds 2r and 2s but limiting the phenyl ring
exposure to the solvent. Actually, such a moiety was
located in a hydrophobic pocket defined by Tyr103,
Tyr108, Ile104, and the Arg100 side chain. Conversely
3s showed its phenyl ring out of this pocket, in a position
similar to that seen for 2r and 2s. The better GST
interaction can be justified also in terms of the inter-
molecular hydrogen bond observed between the 3s
alcoholic group and Tyr108. 4r and 4s reported a
binding mode similar to 3s; in this case, the hydrogen
bond involved the carboxylic group and, respectively,
Arg13 and Tyr108. No specific interactions were re-
ported for the free thiol group of compound 5. In
conclusion, the interaction analysis performed on the
common skeleton of compounds 1-5 compared favorably
to the energy affinity data with the thiol substituent
structures. Such a moiety showed in all cases a similar
binding GST recognition. Variations in the affinity can
be addressed only by the different number and kind of
interactions that the thiol substituent can establish with
GST. It was not a surprise to observe that compound 5
reported the highest binding free energy, followed by
compound 1, which showed the smaller and less func-
tionalized thiol substituent. 2r, 2s, 4r, and 4s reported
a similar binding mode and similar interaction accord-
ing to their ∆Gs. Finally, the highest GST affinity of 3r
and 3s can be attrinuted to additional hydrophobic
effects.
Synthesis. With the aim to synthesize the modeled
molecules 1, 2, 3, and 4 (Figure 1), the relevant
electrophilic precursors were selected, namely 2-bromo-
ethanol (6) for molecule 1, styrene oxide (7) for mol-
ecules 2 and 3, and 2-bromophenylacetic acid (8) for 4.
We have previously developed a mild method for the
one-pot synthesis of S-conjugated cysteines by using a
NaOEt/EtOH system,30 but the poor solubility of GSH
in most organic solvents prevented us from applying the
reported method. We were forced to employ a binary
system with a basic phase-transfer reaction using
tetrabutylammonium hydroxide (TBAH) which is a
strong base and possesses good solubility in water and
in many organic solvents.31 GSH (in the aqueous phase)
reacts with TBAH to form a salt which subsequently
transfers to organic phase for the intrinsic reaction. For
all cases reported here, we used the commercially
available TBAH 40% aqueous solution. Chloroform was
the organic cosolvent selected, based on the reported
higher activity of TBAH in this solvent.32
In a typical reaction, glutathione was dissolved in a
10% aqueous solution of tetrabutylammonium hydrox-
ide and allowed to stir at r.t. with an equal volume of a
4 M solution of the electrophile in chloroform (Scheme
1). Reaction progress was monitored by RP-HPLC
analysis.
It is already known that opening of styrene oxide 7
by means of a nucleophile will produce two different
products.4a In fact, the reaction between GSH and
styrene oxide in basic media gives two regioisomers, 2
and 3, obtained in the ratio 48:52, with a combined
overall yield of 68%. It was possible to separate these
isomers by means semipreparative RP-HPLC as dia-
steromeric mixtures, but efforts to obtain pure dia-
stereoisomers failed. In the case of 2-bromophenylacetic
acid (8), the product 4 was obtained as diastereomeric
mixture with a yield of 65% after semipreparative
RP-HPLC separation. The separation of the pure dias-
tereoisomeric form of compounds 2-4 does not seem a
substantial problem since the ∆G’s evaluation for chiral
substituted compounds 2-4 indicated the configuration
of the thiol side chains to have only weak effects on GST
affinity (Table 2).

6088 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Procopio et al.
Table 2. Yield Obtained after RP-HPLC Separation of
Compounds 1-4
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compound
yield %
1
2
3
4
75
31
34
65
Table 3. Enzymatic Kinetics in Vitroa
compound
IC50 (µM)
Ki (µM)
mechanism
1
2
3
4
280
9.0
4.7
5.5
187
mix
1.11
0.49
0.66
pure competitive
pure competitive
pure competitive
GSH
CDNB
a Kinetic parameters: KM
) 250 µM; KM
) 1.4 mM.
h-GSTP1-1 activity was assayed spectrophotometric-
ally at 340 nm, according to procedure of Habig and
Jakoby. Inhibition experiments were performed using
1 cm cuvettes with 1 mL (final volume) of 0.1 M
potassium phosphate buffer (pH 6.5) containing 1 mM
EDTA, GSH (from 0.1 to 3 mM), 1 mM CDNB (1-chloro-
2,4-dinitrobenzene), suitable amounts of enzyme, and
in the presence of fixed inhibitor (compounds 1-4)
concentrations ranging from 0 to 1 mM.33
The assays were performed using the enzyme
hGSTP1-1 >sp|P09211|GTP-human glutathione S-
transferase P (EC 2.5.1.18) (GST class-pi) (GSTP1-1),
Homo sapiens (human).
The results reported in Table 3 show that 2, 3, and 4
inhibit GST catalytic action through a competitive
mechanism with low IC50 and Ki values, as compared
to many other GST inhibitors proposed. Compound 1,
in contrast, seems to be the only poor GST inhibitor.
Actually, such an observation was previously high-
lighted by molecular modeling simulation that indicated
hydrophobic GRID maps in the area where the thiol-
substituted chain was located by docking experiments.
Therefore, the interaction between GST and compound
1, due to its small hydrophilic side chain, lacks, with
respect to 2-4, a relevant hydrophobic contribution;
however, this result may not reflect other GST classes.
Experimental Section
In a typical reaction, 500 mg (1.63 mmol) of glutathione in
7.0 mL of aqueous n-Bu4NOH 10% and 7.0 mL of a 4 M
chloroform solution of the electrophile were stirred at r.t. The
progress of reaction was followed bu RP-HPLC analysis
[Phenomenex Jupter C18, 250 × 4.6 mm, 10 µm, UV 272 nm,
1.0 mL/min, (H2O (1‰ TFA/methanol 80/20]. The reaction was
completed in almost 36 h. Acidification with trifluoroacetic acid
(TFA) and evaporation under vacuum gave the product which
was purified by semipreparative RP-HPLC chromatography
[Phenomenex Jupter C18, 250 × 10 mm, 10 µm, UV 272 nm,
4.0 mL/min, (H2O (1‰ TFA/methanol 80/20] (see Supporting
Information).
Supporting Information Available: Code list of the 45
GST human isoform models found into PDB citied in Table 1
and Figure 2; Figure 2 reporting ligand structures and their
RMSd in angstoms computed, with respect to the 6GSS, onto
the GSH common skeleton in the conformation bound to the
active site of each PDB models; Table listing yield, elemental
analyses, 1H NMR, and MS data of target compounds 1-4,
and sequence of hGSTP1 used in the biological assay. This
material is available free of charge via the Internet at http://
pubs.acs.org.

Glutathione-S-Transferase Inhibitors
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