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Download by: [Universita G D Annunzio], [Grazia Luisi] Date: 09 September 2015, At: 07:35
Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Nitrobenzoxadiazole-based GSTP1-1 inhibitors
containing the full peptidyl moiety of
(pseudo)glutathione
Grazia Luisi, Adriano Mollica, Simone Carradori, Alessia Lenoci, Anastasia De
Luca & Anna Maria Caccuri
To cite this article: Grazia Luisi, Adriano Mollica, Simone Carradori, Alessia Lenoci, Anastasia
De Luca & Anna Maria Caccuri (2015): Nitrobenzoxadiazole-based GSTP1-1 inhibitors
containing the full peptidyl moiety of (pseudo)glutathione, Journal of Enzyme Inhibition and
Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1070845
Published online: 02 Sep 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
!2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1070845
RESEARCH ARTICLE
Nitrobenzoxadiazole-based GSTP1-1 inhibitors containing the full
peptidyl moiety of (pseudo)glutathione
Grazia Luisi
1
, Adriano Mollica
1
, Simone Carradori
1
, Alessia Lenoci
2
, Anastasia De Luca
3
, and Anna Maria Caccuri
3,4
1
Department of Pharmacy, ‘‘Gabriele d’Annunzio’’ University, Chieti, Italy,
2
Department of Drug Chemistry and Technologies, ‘‘Sapienza’’ University,
Rome, Italy,
3
The NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of Tor Vergata, Rome, Italy, and
4
Department of Experimental Medicine and Surgery, University of Tor Vergata, Rome, Italy
Abstract
Context: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to
overcome drug resistance in oncology practice.
Objective: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding
g-oxa-glutamyl isostere (compounds 1and 5, respectively) have been disclosed as GST
inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD
thioethers.
Materials and methods: Synthesis of derivatives 1and 5and in vitro evaluation on human
GSTP1-1 and M2-2 are reported.
Results: Conjugates 1and 5were found to be low micromolar inhibitors of both isoforms.
Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1
isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol
(NBDHEX).
Discussion and conclusions: Spectroscopic data are congruent with the formation of a stable
sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5is
suitable for in vivo modulation of GST activity in cancer treatment.
Keywords
Glutathione S-conjugates, glutathione
S-transferases, nitrobenzoxadiazole
S-derivatives, pseudopeptide, urethane
bond
History
Received 7 May 2015
Revised 3 July 2015
Accepted 3 July 2015
Published online 1 September 2015
Introduction
The development of resistance to antineoplastic agents remains a
primary concern in cancer therapy. Despite this circumstance, the
comprehension of the multiple mechanisms that contribute to cell
survival following cytotoxic treatment has remarkably evolved.
Thus, alongside the view that makes resistance arising from
events limiting intracellular drug accumulation and target inter-
action, such as minor uptake, enhanced extrusion of the
compound by energy-dependent transporters and/or induction of
chemical-detoxifying mechanisms, recent insights direct on cell
insensitivity to factors that act downstream of the initial drug-
induced insult through the apoptotic machinery.
As a result, a renewed interest has been focused on the
multifunctional family of glutathione S-transferases (GSTs)
1–3
,
since these predominantly cytosolic enzymes were recognized to
play crucial roles in this context
4
. The most widely investigated
function of GSTs is the metabolic conjugation reaction of
electrophilic compounds, including carcinogens and anticancer
drugs, to reduced glutathione (tripeptide g-L-glutamyl-L-cystei-
nyl-glycine, GSH)
5
. It has been shown that different GST
isoenzymes are overexpressed in many cancer cell lines and that
cytosolic GSTs as well as GSH biosynthesis are up-regulated by
electrophiles. According to this evidence, it is still widely
accepted that activation of the cellular GST/GSH system can
contribute directly to drug resistance in some tumor cell types via
its detoxifying activity
6
. More recently, however, a fundamental
non-catalytic, ligand binding activity has been proposed for GST
isoforms alpha, mu and pi which, to a different extent, are able to
associate with the c-Jun N-terminal kinase (JNK) complex
7
.
In addition, tumor necrosis factor receptor-associated factor 2
(TRAF2), an upstream activator of JNK, can be sequestered by
the pi class isoenzyme of GST, glutathione S-transferase P1-1
(GSTP1-1)
8
, thus preventing the MAPK/JNK signaling cascade
which is part of the apoptotic event. Although GSTP1-1 is
overexpressed in a wide range of treated (in addition to untreated)
human solid tumors, its relatively weak affinity for the majority of
anticancer drugs indicates that the mentioned interfering role in
signal transduction is predominant on the catalytic function.
Because of their multi-faced involvement in modulation of cell
survival and stress response
9
, GSTs keep on to be attractive drug
targets for both chemists and biologists, and inhibition of these
enzymes, particularly the P1-1 isoform, represents one of the most
opportune strategy to sensitize tumor tissues to apoptotic and
antiproliferative effects of anticancer drugs.
A variety of GST inhibitors have been reported to date,
including glutathione S-conjugates (GS-R), GSH analogs and
Address for correspondence: Grazia Luisi, PhD, Department of
Pharmacy, ‘‘Gabriele d’Annunzio’’ University, Via dei Vestini 31,
66013 Chieti, Italy. Tel: +39 08713554473. Fax: +39 08713554911. E-
mail: gluisi@unich.it
Anna Maria Caccuri, Professor, Department of Experimental Medicine
and Surgery, University of Tor Vergata, Via Montpellier 1, 00133 Rome,
Italy. Tel: +39 0672596204. E-mail: caccuri@uniroma2.it
Downloaded by [Universita G D Annunzio], [Grazia Luisi] at 07:35 09 September 2015
non-peptide compounds
10–12
. In our long-term research in this
area, we developed the non-glutathione molecule 6-(7-nitroben-
zo[c][1,2,5]-oxadiazol-4-yl)thiohexan-1-ol (NBDHEX), which
inhibits human GSTs at micromolar or submicromolar
concentrations
13
.
Mechanistically, NBDHEX behaves like a mechanism-based
inhibitor of the transferase; after conjugation with GSH it forms a
tetrahedral intermediate (sigma or Meisenheimer complex) which
is strongly stabilized by the GST active site. Tested on several
human cancer cell lines, NBDHEX was shown to possess a high
antiproliferative activity, which depends on its ability to arrest cell
cycle and trigger apoptosis through dissociation of the GSTP1-1
from the complexes with JNK and TRAF2
14–16
. Unfortunately,
NBDHEX seems to be less specific for GSTP1-1 (IC
50
¼0.80 mM)
than for GSTM2-2 (IC
50
¼0.01 mM). Furthermore, its pharmaco-
kinetic profile is hampered by a very low water solubility. Very
recently we designed and synthesized various NBDHEX analogs,
some of which were endowed with greater water solubility than
the parent compound
17
.
In the search for potent and selective inhibitors of GSTs much
attention has been devoted to glutathione S-conjugates, following
the observation that GSTs of the alpha, mu and pi classes are
particularly sensitive to product inhibition
18,19
. It is well noted
that GS-R conjugates use both recognition areas for binding to
GSTs, i.e. the GSH-binding site (the G-site) and the hydrophobic
binding region for the electrophilic substrate (the H-site). Since
the H-site is made up of multiple distinct and partially
overlapping hydrophobic subsites
20
, conjugation with GSH is
considered a valid approach to reduce the mobility of hydrophobic
R groups within this large cavity. Therefore, the linking of a
glutathionyl moiety to the nitrobenzoxadiazole scaffold may
improve the inhibitor selectivity toward GSTP1-1 by exploiting
the differences in both G- and H-binding sites of distinct GST
isoforms.
As a matter of fact, in cells glutathione S-conjugates are
exposed to degradation by g-glutamyl transpeptidase (g-GT),
which selectively cleaves the isopeptidic g-Glu-Cys bond of GSH
and its S-linked derivatives, and to recognition by efflux pumps
belonging to the ATP-binding cassette (ABC) superfamily, such
as the multidrug resistance-associated protein I (MRP1/
ABCC1)
21,22
. MRP1 plays an important role in extruding
cytotoxic drugs from the cell and its overexpression has long
been considered a cause of failure of anticancer chemother-
apy
23,24
. Despite this, it is worthy to note that one of the most
selective GSTP1-1 inhibitor to date is TER 117, the S-benzyl
conjugate of the GSH mimic containing the lipophilic C-terminal
phenylglycyl residue in place of glycine. This peptide competi-
tively inhibits the human transferases with an approximately one
to two orders of magnitude increase in the affinity for GST P1-1
compared with other GST isoforms
10,25
. Furthermore, its diethyl
ester derivative TER199, which is proceeding in phase II clinical
trials as an antimyelodysplastic agent, was shown to act as an
effective inhibitor of the MRPI transporter. The anticancer
potential is therefore augmented as a result of the combined
inhibition of GSTP1-1 activity and MRP1 transport
26
.
For the purpose to obtain further insights on this topic, we were
then interested in investigating 7-nitrobenzoxadiazol-4-yl (NBD)
S-conjugates of the GS-R type. Of necessity we considered some
structural modifications of the GSH peptide moiety to achieve
stability toward g-GT and possibly ameliorate bioavailability. Over
the years we designed a variety of GSH mimics and derivatives
characterized by amino acid substitution and/or bioisosteric
replacements of the g-glutamyl linkage, addressed to enhance
both metabolic resistance and affinity to GST binding sites,
through the attribution of specific chemical and conformational
properties to the modified GSH backbone
27–34
. Among these, the
g-oxa-glutamyl (Glo) analog of GSH, H-Glo[Cys-Gly-OH]-OH,
deserves particular attention for more than one reason: (1) as
expected, the replacement of the scissile g-glutamyl-cysteinyl
peptide bond with the OCONH unit assures resistance to g-GT-
mediated hydrolysis
29,35
; (2) the substitution does not sensibly
alter the physico-chemical properties of the tripeptide; (3) groups
crucial for binding to the G-site resemble those of the natural
ligand, with the H-bond donor/acceptor potential of the GSH
backbone fairly maintained and (4) more importantly, S-conjugates
of this urethane mimic of glutathione have been shown to inhibit
MRP1
36
.
Thus we report here a straight preparation of the glutathione
conjugate H-Glu[Cys(NBD)-Gly-OH]-OH (GS-NBD, 1) and the
synthesis of its new analog H-Glo[Cys(NBD)-Gly-OH]-OH or
(OCONH) GS-NBD (5), to be comparatively evaluated as
inhibitors of human GSTP1-1 and M2-2.
The structural relationship between compounds 1,5and
NBDHEX is evident (Figure 1): this latter may be considered as a
molecular simplification, lacking the polar and H-bond donor/
acceptor groups, of the Cys(NBD)-Gly moiety obtained from
removal of the (pseudo)glutamyl recognition determinant that
characterizes the full peptide conjugates.
A further interesting feature of these ligands lies in their
fluorescent properties that enable indicative spectral character-
ization and monitoring in biological media.
Methods
Chemistry
S-conjugates 1and 5were synthesized in good overall yields
employing solution phase procedures, as outlined in Schemes 1
and 2. A minor problem associated with the preparation of the
target peptides was the chemical (and photochemical) instability
of 4-chloro-7-nitrobenzo[c][1,2,5]-oxadiazole (NBD-Cl), there-
fore the arylation reactions were preferably conducted with the
exclusion of light. Compound 1was easily prepared by treating an
hydroalcoholic solution of glutathione with NBD-Cl in the
presence of pyridine (2.5 equiv.) to keep the pH value at 5.5
(Scheme 1). In these conditions, reaction was complete in 1h and
resulted in a pure and abundant crop of the expected S-heteroaryl-
conjugate 1as a yellow precipitate. When sodium phosphate
buffer (pH 7.0) was used as an alternative medium, the formation
of 1proceeded with lengthy times.
The strategy to the g-oxa-glutamyl analog 5required of neces-
sity to assemble the whole tripeptide first, performing the
S-arylation in the final step. The pseudopeptide was prepared
essentially after our reported protocol, with the urethanic junction
NBDHE
X
CYSTEINYL S-NBD CONJUGATES OF GSH AND PSEUDO-GSH
X
OH
S
N
O
N
NO2
NO2
N
O
N
S
N
H
OH
O
O
N
H
OO
HO
NH2
Figure 1. Backbone-side chain structural analogies connecting NBDHEX
and the dipeptide unit in conjugates 1(X ¼CH
2
) and 5(X ¼O).
2G. Luisi et al. J Enzyme Inhib Med Chem, Early Online: 1–7
Downloaded by [Universita G D Annunzio], [Grazia Luisi] at 07:35 09 September 2015
made up by convenient functionalization of a protected serine
residue (Scheme 2)
29
. By following the aforementioned conditions
for heteroaryl substitution, the S-conjugate was collected in only
moderate yields, together with significative amounts of non-
reacted starting thiol. Attempts to force the S
N
Ar toward comple-
tion in phosphate buffer were successful by using a stoichiometric
excess of pseudoglutathione and longer reaction times.
Final compounds were purified to apparent homogeneity by gel-
permeation chromatography and fully characterized by
1
H and
13
C
NMR spectroscopy. Spectral data for both compounds 1and 5are
congruent with the expected structures of NBD conjugates at the
sulfur atom and exclude the formation of isomeric N-derivatives
due to the presence of a second nucleophilic group in the reactant.
This result is in accord with previous note reporting that, differ-
ently from cysteine S-NBD derivative, which can easily undergo
S!Naryl transfer, glutathione forms only the S-conjugate
37
.
Biological assays
Peptide conjugates 1and 5were assayed in vitro for
their ability to inhibit the GSH conjugation reaction with
1-chloro-2,4-dinitro-benzene (CDNB), mediated by human GST
P1-1 and M2-2. Inhibition experiments were performed at 25 C
in 0.1 M potassium phosphate buffer pH 6.5 containing constant
(1 mM) GSH and acceptor substrate concentrations and variable
amounts of the inhibitors.
Experimental
Peptide synthesis
GSH and amino acid derivatives were purchased from Sigma-
Aldrich (St. Louis, MO) and Bachem (Bubendorf, Switzerland).
All other chemicals and solvents were of analytical grade and
were supplied from Sigma-Aldrich and VWR (Radnor, PA). All
the reactions were monitored by analytical TLC on Merck
(Kenilworth, NJ) 60 F
254
plates developed with the following
solvents: (a) n-BuOH/AcOH/H
2
O (2:1:1); (b) CHCl
3
/MeOH
(99:1); (c) CHCl
3
/MeOH (98:2). Column chromatography was
carried out in absorption using Merck 60 silica gel (230–400
mesh). Melting points were determined on a Bu
¨chi B-450
apparatus (Uster, Switzerland) and are uncorrected. Elemental
analyses (C, H, N and S) were performed on a Carlo Erba 1106
Analyzer (Milano, Italy) and were within ±0.4% of the theoretical
values. IR spectra were recorded employing a Perkin-Elmer FTIR
1600 spectrophotometer (Waltham, MA).
1
H (300 MHz) and
13
C
(75.43 MHz) NMR spectra were acquired on a Varian VXR-300
instrument (Palo Alto, CA). Chemical shifts are reported in ppm,
referenced to residual solvent peaks and multiplicities are indicated
as s (singlet), d (doublet), t (triplet), m (multiplet) and br (broad).
Peak assignments were confirmed by 2D
1
H–
13
C hetero-correlated
experiments.
H-Glu[Cys(NBD)-Gly-OH]-OH (1)
To a stirred solution of GSH (0.31 g, 1.0 mmol) in a (2:1) mixture
of H
2
O/EtOH (6 mL) solid NBD-Cl (0.20 g, 1.0 mmol) and Py
(0.2 mL, 2.5 mmol) in EtOH (1 mL) were added at room
temperature. The initial solution was kept under vigorous stirring
with the exclusion of light for 1 h, during which time pH
was maintained to 5.5 by addition of Py. The reaction mixture
was diluted with EtOH and filtered in vacuo. The precipitate
was recrystallized from acetone/EtOH (2:1) to give 0.33 g of
compound 1as a yellow powder (70% yield). R
f
(a) ¼0.4; m.p.
(Ref.29)
5
Trt
Trt
a
c-f
b
2
4
3
Fmoc
Trt
Boc
But
But
But
But
But
Boc
S
O
N
H
O
OH2N
NH2
NO2
O
O
N
H
O
S
N
H
NpO
O
O
N
H
OO
N
H
ON
H
N
H
O
O
O
S
O
OO
N
O
N
S
N
H
OH
O
O
N
H
O
O
OH
O
Scheme 2. Synthesis of H-Glo[Cys(NBD)-Gly-OH]-OH (5). Reagents and conditions: (a) DBU, DCM, r.t., 15 min; (b) dioxane, 80 C, 20 h; (c) I
2
,
MeOH, r.t., 4 h; (d) TFA, r.t., 4 h; (e) P(nBu)
3
, aq. NH
3
,n-PrOH/H
2
O, r.t., 1 h; (f) NBD-Cl, phosphate buffer pH 7.0, r.t., 48 h, obscured.
SH
N
H
OH
O
O
N
H
O
HO
NH2
O
NO2
N
O
N
S
N
H
OH
O
O
N
H
O
GSH
GS-NBD (I)
HO
NH2
O
Scheme 1. Synthesis of GS-NBD (1). Reagents and conditions: NBD-Cl,
Py, H
2
O/EtOH, r.t., 1 h, obscured.
DOI: 10.3109/14756366.2015.1070845 Nitrobenzoxadiazole-based GSTP1-1 inhibitors 3
Downloaded by [Universita G D Annunzio], [Grazia Luisi] at 07:35 09 September 2015
199–200 C (dec.). IR (KBr)
max
: 3350, 3055, 1690, 1645, 1510,
1335 cm
1
;
1
H NMR ([D
6
]DMSO): 1.7–2.0 (2H, m, Glu
b-CH
2
), 2.2–2.4 (2H, m, Glu g-CH
2
), 3.15 (1H, m, Glu a-CH), 3.4
(1H, m, Cys b-CH
B
), 3.7 (2H, m, Gly CH
2
), 3.75 (1H, m, Cys
b-CH
A
), 4.85 (1H, m, Cys a-CH), 7.6 (H, d, J¼7.5 Hz, hetArH),
8.6 (1H, d, J¼7.5 Hz, hetArH), 8.8 (1H, d, J¼8.2 Hz, Cys NH),
8.95 (1H, t, J¼5.0 Hz, Gly NH).
13
C NMR ([D
6
]DMSO): 27.25
(Glu Cb), 31.96 (Glu Cg), 33.68 (Cys Cb), 41.98 (Gly Ca), 51.75
(Cys Ca), 53.62 (Glu Ca), 123.26, 133.08, 139.79, 143.32, 149.84
(heteroaromatics), 170.42, 171.25, 171.55, 172.73 (CO). Anal.
calcd for C
16
H
18
N
6
O
9
S: C, 40.85; H, 3.86; N, 17.87, S, 6.82.
Found: C, 40.68; H, 3.70; N, 18.05, S, 6.98.
Fmoc-Cys(Trt)-Gly-OtBu (2)
Fmoc-Cys(Trt)-OH (1.2 g, 2.1 mmol) was suspended in THF
(5 mL) and HOBt (0.28 g, 2.1 mmol) was added under stirring.
The solution was cooled to 0 C and an ice-cold solution
containing HCl . H-Gly-OtBu (0.35 g, 2.1 mmol) and NMM
(0.21 g, 2.1 mmol) in THF (5 mL) was added, followed by
portionwise addition of a solution of DCC (0.43 g, 2.1 mmol) in
THF (4 mL). After 6 h at 0 C and 16 h at 5 C, the reaction
mixture was filtered and the resulting solution evaporated under
reduced pressure. The residue was taken up in AcOEt and the
organic layer washed with 1 N KHSO
4
, saturated aqueous
NaHCO
3
and H
2
O. The residue obtained after drying and solvent
evaporation was chromatographed on silica gel using a CHCl
3
/
MeOH (98:2) mixture as eluent, to give 2(1.4 g, 94%) as a white
foam. R
f
(b) ¼0.5; IR (neat)
max
: 3410, 1730, 1670 cm
1
;
1
H NMR (CDCl
3
): 1.4 (9H, s, CH
3
), 2.55 (1H, m, Cys b-CH
B
),
2.7 (1H, m, Cys b-CH
A
), 3.2 (1H, m, Cys a-CH), 3.75 (2H, m,
Gly CH
2
), 4.2 (1H, m, Fmoc CH), 4.35 (2H, m, Fmoc CH
2
), 6.0
(1H, br d, Cys NH), 7.2–7.4 (15H, m, ArH), 7.5 (1H, br t, Gly
NH). Anal. calcd for C
43
H
42
N
2
O
5
S: C, 73.90; H, 6.06; N, 4.01; S,
4.59. Found: C, 74.12; H, 6.15; N, 3.97; S, 4.46.
H-Cys(Trt)-Gly-OtBu (3)
The protected dipeptide 2(0.56 g, 0.80 mmol) was dissolved in
DCM (3 mL) and DBU (0.12 g, 0.80 mmol) in DCM (1 mL)
34
was
added in portions at room temperature. After 15 min the solvent
was evaporated to dryness and the residue eluted from a silica gel
column using a CHCl
3
/MeOH (97:3) mixture as eluent, to give
compound 3as an oil (0.34 g, 89%). R
f
(c) ¼0.8; IR (neat)
max
:
3360, 1730, 1650 cm
1
;
1
H NMR (CDCl
3
): 1.45 (9H, s, CH
3
),
2.6 (1H, m, Cys b-CH
B
), 2.8 (1H, m, Cys b-CH
A
), 3.0 (1H, m,
Cys a-CH), 3.8 (2H, m, Gly CH
2
), 7.2-7.45 (15H, m, ArH), 7.5
(1H, br t, Gly NH). Anal. calcd for C
28
H
32
N
2
O
3
S: C, 70.56; H,
6.77; N, 5.88, S, 6.73. Found: C, 70.31; H, 6.91; N, 5.65, S, 7.04.
Boc-Glo[Cys(Trt)-Gly-OtBu]-OtBu (4)
To a stirred solution of dipeptide ester 3(3.3 g, 7.0 mmol) in
dioxane (10 mL) Boc-Glo(ONp)-OtBu
29
(3.0 g, 7.0 mmol) in
dioxane (5 mL) was added. The mixture was warmed to 80 C
and left to stir for 20 h. The solution was evaporated under
vacuum to give an oily residue which was taken up in CHCl
3
. The
organic layer was washed with 0.5 N HCl, saturated aqueous
Na
2
CO
3
and H
2
O, dried and filtered. The filtrate was evaporated
under reduced pressure, and the residue chromatographed on a
silica gel column using a CHCl
3
/MeOH (98:2) mixture as eluent,
to give the protected tripeptide 4as a white foam (3.8 g, 70%). R
f
(c) ¼0.5; IR (KBr)
max
: 3400 br, 3100, 1735, 1705, 1660,
1540 cm
1
;
1
H NMR (CDCl
3
): 1.4 (27H, s, CH
3
), 2.65 (1H, m,
Cys b-CH
B
), 2.85 (1H, m, Cys b-CH
A
), 3.05 (1H, m, Gly CH
B
)
3.2 (1H, m, Gly CH
A
), 4.1 (2H, m, Cys and Glo a-CH), 4.3 (2H,
m, Glo b-CH
2
), 5.65 (1H, br t, Glo NH), 6.6 (1H, br d, Cys NH),
7.2–7.35 (15H, m, ArH), 7.5 (1H, br t, Gly NH). Anal. calcd for
C
41
H
53
N
3
O
9
S: C, 64.46; H, 6.99; N, 5.50, S, 4.20. Found: C,
64.24; H, 7.05; N, 5.72, S, 4.31.
H-Glo[Cys(NBD)-Gly-OH]-OH (5)
Compounds {Boc-Glo[Cys-Gly-OtBu]-OtBu}
2
, {H-Glo[Cys-Gly-
OH]-OH}
2
and H-Glo[Cys-Gly-OH]-OH were prepared by
following the already published protocol
29
, and characterized
by NMR spectroscopy.
H-Glo[Cys-Gly-OH]-OH (0.42 g, 1.3 mmol) was dissolved in
0.1 M sodium phosphate buffer (pH 7.0) (4 mL) under stirring at
room temperature before the addition of solid NBD-Cl (0.13 g,
0.65 mmol). After 48 h with the exclusion of light, dur ing which
time the reaction course was followed by TLC, the resulting
suspension was evaporated under reduced pressure and the crude
material taken up in H
2
O. Recrystallization from H
2
O/MeOH
afforded conjugate 5as a powdery yellow solid (0.37 g, 60%). R
f
(a) ¼0.45; IR (KBr)
max
: 3320 br, 3050, 1690–1615, 1510,
1335 cm
1
;
1
H NMR (D
2
O): 2.8 (1H, m, Cys b-CH
B
), 3.1 (1H,
m, Cys b-CH
A
), 3.6 (1H, m, Gly CH
2
), 3.8 (2H, m, Glo and Cys
a-CH), 4.2 (2H, m, Glo b-CH
2
), 7.4 (1H, d, J¼7.0 Hz, hetArH),
8.4 (1H, d, J¼7.0 Hz, hetArH).
13
C NMR (D
2
O): 33.84 (Cys
Cb), 41.59 (Gly Ca), 51.43 (Cys Ca), 54.02 (Glo Ca), 67.92 (Glo
Cb), 123.59, 133.03, 139.53, 143.37, 149.91 (heteroaromatics),
156.84, 174.86, 176.74 (CO). Anal. calcd for C
15
H
16
N
6
O
10
S: C,
38.14; H, 3.41; N, 17.79, S, 6.79. Found: C, 38.36; H, 3.48; N,
17.63, S, 6.80.
GST inhibition
Human GSTM2-2 and GSTP1-1 were expressed in Escherichia
coli and purified as previously described
38
. The GST activity was
measured in 0.1 M potassium phosphate buffer (pH 6.5) contain-
ing 1 mM GSH, 1 mM CDNB as co-substrate and 0.1 M EDTA
39
.
Inhibition experiments were performed by adding variable
amounts of either GS-NBD 1or its analog 5, ranging from 0.1
to 50 mM, to the assay mixture. IC
50
is defined as the inhibitor
concentration which fulfils 50% of catalytic activity inhibition.
IC
50
values were obtained by the best fit of the experimental data
to a hyperbolic binding equation. The selectivity index (SI)
toward GSTP1-1 is calculated from the IC
50 GSTP1-1
/IC
50 GSTM2-2
ratio, therefore a decrease of SI compared with that of NBDHEX
(SI ¼80) indicates an increase of compound selectivity toward
GSTP1-1. Activity was recorded at 340 nm, where the NBD
conjugate absorbs (e¼9.6 mM
1
cm
1
); the UV–vis spectrum of
GS-NBD (1) (50 mM), in 0.1 M potassium phosphate buffer pH
7.0, was recorded, before and after the addition of either 1 mM
GSH or stoichiometric amounts of GSTP1-1 and 1 mM GSH,
using a Kontron double-beam Uvikon 940 spectrophotometer
(Redwood City, CA) thermostatically operating at 25 C.
Results
Conjugate 1inhibits GSTP1-1 and GSTM2-2 with IC
50
values
of 7.8 and 0.26 mM, respectively (Figure 2, panels A and B). The
inhibitory profile of pseudoglutathione derivative 5on the target
enzymes is very similar, with IC
50
values of 7.9 and 0.3 mMfor
the GSTP1-1 and the GSTM2-2 isoforms (panels C and D,
respectively, in Figure 2).
Table 1 collects data for GST inhibition by compounds 1and 5
together with representative conjugates reported so far. Inhibitors
are listed in descending order of activity toward GSTP1-1,
starting from the most potent (and selective) prototypical inhibitor
TER 117.
The analysis of data allows the following considerations: apart
from TER 117, the compound in the series that shows the
4G. Luisi et al. J Enzyme Inhib Med Chem, Early Online: 1–7
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strongest selectivity for the target enzyme with respect to the
M2-2 isoform is GS(Hex). However, the latter is a less efficient
GSTP1-1 inhibitor compared to a range of compounds, including
NBDHEX and the present conjugates 1and 5. Despite inhibitors 1
and 5show a tenfold decrease in potency with respect to
NBDHEX against the target isoform GSTP1-1, yet the affinity of
both peptides for the GSTM2-2 isozyme is even lower; thus the
loss in inhibitory activity toward GSTP1-1 is counterbalanced by
an almost threefold gain in selectivity toward the same isoform, as
evidenced by their SI index (as defined above) (values of 30 and
27 for compounds 1and 5, respectively, compared with the
NBDHEX value of 80). Interestingly, activity data obtained for
the two inhibitors are superimposable. The significance of these
results is discussed in the next session.
In analogy with the mechanistic behavior of many nitro-
substituted aromatic compounds
41
, the inhibition mechanism for
both NBD derivatives 1and 5implies that they bind to the GST
active site and form a sigma-complex intermediate with the
resident GSH. In fact, the UV–vis spectrum of GS-NBD is
minimally affected by the presence of 1 mM GSH (Figure 3,
dashed line). However, the spectral perturbation is significantly
higher when GS-NBD is incubated with both GSH and GSTP1-1
(Figure 3, solid line). In this case, the band of GS-NBD, centered
at 419 nm, almost disappears and a new absorption band appears
at about 348 nm.
It is worth mentioning that the two recently reported GSTP1-1
inhibitors Z-Cys(NBD)-OMe and Boc-Cys(NBD)-OMe (Table 1),
also featuring an albeit minimal peptide nature, share with 1and 5
the ability to form a sigma-complex inside the active cleft of the
transferase; however, to our knowledge, the present NBD
S-conjugates 1and 5represent the first examples of mechan-
ism-based inhibitors for the human P1-1 isoform characterized by
(A) (B)
(C) (D)
Figure 2. Effects of NBD S-conjugates 1and 5on the catalytic activity of GSTP1-1 and GSTM2-2. The GST activity was reported as percent of GST
activity inhibition. The solid line is the best fit of the experimental data to a hyperbolic binding equation which fulfils the IC
50
values of 7.8 ± 1.3 and
0.26 ± 0.02 mM for the reaction catalyzed by GSTP1-1 (panel A) and GSTM2-2 (panel B), respectively, in the presence of GS-NBD 1as inhibitor.
When the enzymatic activity was measured in the presence of NBD conjugate 5, the IC
50
values found were 7.9 ± 0.8 and 0.29 ± 0.04 mM for GSTP1-1
(panel C) and GSTM2-2 (panel D), respectively. Data represent means ± SD of three independent experiments.
Table 1. Inhibition of human GSTP1-1 and GSTM2-2 isoforms by compounds bearing a peptide, non-peptide or pseudopeptide skeleton.
Compound GSTP1-1 (IC
50
,mM) GSTM2-2 (IC
50
,mM) SI index (IC
50
GSTP1-1/IC
50
GSTM2-2) Refs.
TER 117
a
0.4* 184* 0.002
10,25
Z-Cys(NBD)-OMe
a
0.4 0.008 50
17
Boc-Cys(NBD)-OMe
a
0.6 0.005 120
17
NBDHEX
b
0.8 0.01 80
13
1
a
7.8 0.26 30 Present study
5
c
7.9 0.30 26 Present study
GS(Hex)
a
10.0 36.0 0.3
10
GS(Fm)
a
32.9 6.5 5
32
GS-EA
a
11 50.1 4110
40
*Determined as K
i
values.
a
Peptide,
b
non-peptide or
c
pseudopeptide skeleton.
DOI: 10.3109/14756366.2015.1070845 Nitrobenzoxadiazole-based GSTP1-1 inhibitors 5
Downloaded by [Universita G D Annunzio], [Grazia Luisi] at 07:35 09 September 2015
a full (pseudo)glutathione skeleton. Thus, 1and 5deserve
attention among peptide inhibitors, since their binding mechanism
to target enzyme is alternative with regard to TER 117.
Discussion
We have previously reported a series of NBD thioether derivatives
as nano/micromolar inhibitors of GSTs, a family of detoxifying
enzymes also involved in cancer drug resistance phenomenon.
It has been evidenced that the prototype thioether NBDHEX
promotes GSTP1-1 dissociation from the GSTP1-1/JNK hetero-
complex, resulting in cell death by apoptosis
14
. A detailed
investigation of the interaction between NBDHEX and different
human GST isoenzymes revealed that this molecule behaves like
a mechanism-based inhibitor: in fact, it is conjugated with GSH in
the GST active site leading to a stable sigma-complex that
represents the real inhibitor species
13
.
In this work we tested the inhibitory properties on GSTP1-1
and GSTM2-2 of NBD thioethers 1and 5, in which the aliphatic
chain of 6-mercapto-hexanol has been replaced by the peptidyl
moiety of GSH and its g-oxa-analog, respectively. Compounds 1
and 5are good inactivators of both isoforms, with IC
50
s in the low
micromolar range. As previously observed with NBDHEX
13
, the
GST isoenzyme M2-2 shows much higher sensitivity toward the
inhibitory effect of both derivatives compared to the P1-1 isoform.
It is interesting to note, however, that, despite inhibitors 1and 5
inhibit both GSTs to a lesser extent in comparison with
NBDHEX, they display a threefold reduction in selectivity for
the M2-2 isozyme over GSTP1-1 compared with the aforemen-
tioned inhibitor.
Moreover, the present data reveal that replacement of a peptide
unit with a urethane bond does not affect at all the inhibition
properties of the NBD conjugate; in fact, identical IC
50
values are
found for GS-NBD 1and thioether analog 5.
The inhibition mechanism of conjugates 1and 5suggests that
the main binding determinant for the interaction with the GST
active site is the benzoxadiazole ring of NBD, while the peptidyl
portion of the inhibitor may be easily displaced from the G-site by
free GSH. This is followed by the deprotonation/activation of
GSH, enabling the nucleophilic attack of GSH on the C4 of NBD
to form a sigma-complex. This hypothesis is confirmed by the
UV–vis spectra of GS-NBD, showing that the sigma complex
intermediate is mainly formed into the GST active site. This
evidence, obtained for the first time with NBDHEX
13
, has been
recently confirmed with all compounds bearing substituted linear
or branched alkyl chains at the C4-sulfur atom
17
.
Moreover, the evidence that the NBD- derivative 5may fully
replace GS-NBD 1, may have important effects in cytotoxicity
experiments performed with tumour cell lines, increasing the
intracellular stability of this powerful GST inhibitor.
Conclusion
The present study reports synthesis and biological evaluation of
glutathione and pseudoglutathione nitrobenzoxadiazole S-conju-
gates as peptide inhibitors of human P1-1 and M2-2 glutathione
transferases.
Activity data show that the tested compounds are good
inactivators of both isoforms, with IC
50
s in the micromolar range.
Furthermore, the selectivity profile of compounds 1and 5, albeit
invariantly shifted in favor of the mu isoform, indicates a
significant decrease in affinity for this isozyme in comparison
with the prototype inhibitor NBDHEX.
The backbone-modified analog 5, which represents the g-GT
resistant mimic of 1, combines resistance to g-GT-mediated
hydrolysis with the potential to behave as a dual targeting agent
for GSTP1-1 and MRP1 export pump. Therefore, analog 5may be
suitable for the treatment of drug resistant tumors characterized
by an elevated expression of both GSTP1-1 and MRP1.
Declaration of interest
The authors report no declaration of interest. This study was supported
by grants from Ministry of Education, University, and Research of Italy
(University ‘‘Gabriele d’Annunzio’’, ex 60% 2014, Luisi G.) and from
Associazione Italiana per la Ricerca sul Cancro (AIRC), project IG-10598
(Caccuri A.M.)
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