Role of glutathione transferases in the mechanism of brostallicin activation

Article abstract of DOI:10.1021/bi901689s

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels

Full text of DOI:10.1021/bi901689s

226 Biochemistry 2010, 49, 226–235
DOI: 10.1021/bi901689s
Role of Glutathione Transferases in the Mechanism of Brostallicin Activation
Silvia Pezzola,‡ Giovanni Antonini,§ Cristina Geroni, Italo Beria, Maristella Colombo, Massimo Broggini,^ Nicola
Mongelli, Loris Leboffe,§ Robert MacArthur,@ Alessia Francesca Mozzi,# Giorgio Federici,r and Anna Maria Caccuri*,‡
‡Department of Chemical Sciences and Technologies, University of “Tor Vergata”, Rome, Italy, §Department of Biology, University of
“RomaTre”, Rome, Italy, Nerviano Medical Sciences Srl, Oncology, Nerviano, Italy, ^Laboratory of Molecular Pharmacology,
Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy, @Systems Medicine LLC, a wholly owned subsidiary of
Cell Therapeutics Inc., Seattle, Washington, #Laboratory of clinical biochemistry, TorVergata’s Hospital, Rome, Italy, and
rChildren’s Hospital, Istituto di Ricovero e Cura a Carattere Scientifico ‘‘Bambin Gesuꢀ’’, Rome, Italy
Received September 28, 2009; Revised Manuscript Received December 1, 2009
ABSTRACT: Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising
anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison
with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly
enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-π or GST-μ.
Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and
GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here
indicate that brostallicin binds reversibly to both isoenzymes with Kd values in the micromolar range (the
affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze
the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and
by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is
slowly converted into the final products. This intermediate, identified as the R-chloroamido derivative of the
GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active
form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1,
has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is
provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the
treatment of tumor overexpressing the pi or mu class GST.
Brostallicin 1 (Figure 1) is an anticancer agent under evaluation
in phase I and II clinical trials (1). This synthetic compound is a
DNA minor groove binding drug characterized by an R-bromoa-
crylamido moiety tethered to a distamycin-like frame (Figure 1).
In vitro and in vivo experiments show that it is active against a
variety of experimental murine and human tumors when used as a
single agent. It also displays synergistic activity when combined
with either mechanistically classical or novel molecular-targeted
drugs (2, 3). Brostallicin circumvents tumor resistance mechani-
sms known to inactivate alkylating agents, platinum derivatives,
and camptothecins, maintains efficacy in mismatch repair-
deficient cells, and is very active in inducing apoptosis (3-7).
Even though brostallicin and its chloroacrylamido derivative 2
(Figure 1) have a significant cytotoxic effect on tumor cells,
brostallicin appears unreactive in classical DNA alkylation
assays (8). Therefore, it has been hypothesized that the drug
may be activated by an intracellular reactive nucleophilic species
that could perform a first-step Michael-type attack on the double
bond, forming a reactive adduct able to bind covalently to the
minor groove of DNA (9). Furthermore, experimental evidence
suggests that human glutathione transferases (GSTs,1 EC
2.5.1.18) are essential for the activation of brostallicin. In fact,
the human mu and pi class GST isoenzymes decrease the half-life
of brostallicin in solution (4). This drug was also found to be
significantly more active on tumor cell lines overexpressing the pi
class isoenzyme GSTP1-1 (3, 10). Accordingly, in vivo experi-
ments show that cancer cells overexpressing GSTP1-1, due to
transfection of GSTP1-1 cDNA, are more sensitive to brostallicin
in comparison with tumor cells with lower GSTP1-1 levels (3, 10).
Thus, brostallicin has been hypothesized to be a GST-activated
pro-drug, i.e., a molecule that exploits GST catalytic power for
activation into a cytotoxic agent. In this study, we confirm this
hypothesis by highlighting the role of different GST isoenzymes in
the brostallicin activation mechanism. We used multiple and
distinct experimental approaches to study in detail the nature of
the interaction of brostallicin with GSTP1-1 and GSTM2-2, the
most abundant GST isoenzymes in human tumor cells (11, 12).
The obtained data lead to the conclusion that both GSTs catalyze
the formation of a transient R-chloroamido derivative of the
GSH-brostallicin adduct. We further analyzed the reactivity of
brostallicin and its R-chloroamido derivative in a DNA alkylation
assay, reaching the conclusion that the transient derivative could
represent the active form of the drug.
*To whom correspondence should be addressed: Department of
Chemical Sciences and Technologies, University of “Tor Vergata”,
Via della Ricerca Scientifica, 00133 Rome, Italy. Telephone: 39-
0672596204. Fax: 39-0672594328. E-mail: caccuri@uniroma2.it.
1Abbreviations: GST, glutathione transferase; GSH, glutathione;
CDNB, 1-chloro-2,4-dinitrobenzene; TFA, trifluoroacetic acid; HPLC,
high-performance liquid chromatography; MS, mass spectrometry;
Taq, Thermophilus aquaticus; PCR, polymerase chain reaction.
EXPERIMENTAL PROCEDURES
Materials. 1-Chloro-2,4-dinitrobenzene (CDNB) and GSH
were obtained from Sigma Chemical Co. Brostallicin 1 (PNU-
166196) and its debrominated derivative 3 were synthesized at Ner-
viano Medical Sciences Srl (Nerviano, Italy). Brostallicin-GSH
r
pubs.acs.org/Biochemistry
Published on Web 12/01/2009
2009 American Chemical Society

Article
Biochemistry, Vol. 49, No. 1, 2010 227
F
IGURE 1: (A) Structure of brostallicin (PNU-166196) (1) and analogues. (B) Structure of the brostallicin-GSH adduct (4) and derivatives.
adducts 6 and 7 were generated after a prolonged incubation (∼3 h)
of brostallicin (100 μM) with GSH (1 mM) and GSTP1-1 (1 μM) in
either 0.1 M potassium phosphate buffer (pH 6.5) to yield a mixture
of compounds 6 and 7 or aqueous solution (no buffer), adjusting
the acid solution to pH 6.5 with diluted NaOH, to yield only
compound 6.
sequence as reported by Gill et al. (17) and confirmed by the
bicinchoninic acid method (Pierce). Enzyme molarity was calcu-
lated referring to a single subunit. Molecular masses of 23 and
25.6 kDa per GST subunit were used in the calculations for
GSTP1-1 and GSTM2-2, respectively.
GST Inhibition Experiments. The interaction between the
GSTs and brostallicin was determined, at 25 ꢀC and pH 6.5, by
measuring the decrease in GST activity in the presence of diffe-
rent concentrations of drug (between 0 and 50 μM with GSTP1-1
and between 0 and 1 μM with GSTM2-2). At each brostallicin
concentration, either the CDNB or the GSH concentration was
varied from 0.25 to 2 mM, while the cosubstrate concentration
was maintained constant at 1 mM. Initial velocity data were
reported as double-reciprocal plots. Apparent inhibition con-
stants (Ki) were calculated by replot analysis of the primary
reciprocal plots (18). The interaction of the GSTs with compound
3 (from 0 to 50 μM), 6 (from 0 to 20 μM), or a mixture of 6 and 7
(from 0 to 20 μM) was evaluated by measuring the decrease in
GST activity at pH 6.5, 25 ꢀC, and fixed GSH and CDNB
concentrations (1 mM). IC50 values (the concentration of in-
hibitor that gives 50% of the reaction rate of the control reaction)
were derived from the fit of data to a simple binding isotherm.
HPLC-UV/MS Evidence of the Reaction between GSH
and Brostallicin. The time course of the GST-catalyzed reaction
between GSH and brostallicin was followed by using a HPLC-
UV/MS technique. In a typical experiment, brostallicin (100 μM)
and GSH (1 mM) were incubated at 25 ꢀC in 0.1 M potassium
phosphate buffer (pH 6.5, adjusted to pH 6.5 with diluted HCl)
containing either 1 μM GSTP1-1 or 1 μM GSTM2-2. Alterna-
tively, the same reaction was performed in 0.1 M sodium acetate
buffer (pH 6.5). Aliquots were taken at the indicated times, and
the reaction was stopped by addition of TFA (final concentration
of 1%). HPLC-UV/MS analyses of samples (20 μL) were
Cell Lines and Drug Sensitivity. The MCF-7 human breast
carcinoma cell line and its clones overexpressing the human GST-
π (MCF-7π), GST-μ (MCF-7μ), or GST-R (MCF-7R) gene were
obtained and grown as reported previously (13, 14). All the clones
overexpress the specific transfected isoform, and they have an
increased GST activity, measured using CDNB as the substrate,
over parental cells (13, 14). All cell lines were maintained at 37 ꢀC
and 5% CO2 in medium supplemented with 10% fetal calf serum.
Drug-induced cytotoxicity was determined by a intracellular
ATP monitoring system (CellTiterGlo-Promega) using the En-
vision (PerkinElmer) as a reader. Exponentially growing cells
were seeded and exposed to various drug concentrations. The
antiproliferative activity of brostallicin was evaluated by com-
paring treated versus vehicle-treated control cells using Assay
Explorer (MDL). The dose inhibiting cell growth by 50% (IC50)
was calculated using a sigmoidal interpolation curve.
Enzymes. Recombinant GSTP1-1 and GSTM2-2 were ex-
pressed in Escherichia coli and purified as previously des-
cribed (15). GST activity was measured at 25 ꢀC in 1 mL (final
volume) of 0.1 M potassium phosphate buffer (pH 6.5) contain-
ing EDTA (0.1 mM), GSH (1 mM), and CDNB (1 mM) as a
cosubstrate. The activity was followed spectrophotometrically
at 340 nm, where the GS-DNB reaction product absorbs (ε =
9.6 mM-1 cm-1) (16). Protein concentrations were calculated
from the absorbance at 280 nm assuming ε1mg/mL values of 1.30
and 1.48 for GSTP1-1 and GSTM2-2, respectively. The extinc-
tion coefficient was calculated on the basis of the amino acid

228 Biochemistry, Vol. 49, No. 1, 2010
Pezzola et al.
conducted by combining the ion trap MS instrument with the
SSP4000 HPLC system (Thermo Separation Products) equipped
with an LC Pal autosampler (CTC Analytics) and a UV6000LP
diode array detector (UV detection from 215 to 400 nm).
Instrument control, data acquisition, and processing were per-
formed by using Xcalibur version 1.2 (Finnigan). HPLC was
conducted at room temperature, with a 1 mL/min flow rate, using
a Waters XBridge RP 18 column (4.6 mm ꢀ 50 mm, 2.5 μm).
Mobile phase A consisted of ammonium acetate 5 mM buffer
(pH 5.5 with acetic acid) and acetonitrile (90:10), and mobile
phase B consisted of ammonium acetate 5 mM buffer (pH 5.5
with acetic acid) and acetonitrile (10:90); the gradient was from 0
to 100% B over 7 min then 100% B for 2 min before re-
equilibation. The ion trap spectrometer was operated in the full
scan positive ion mode, in a range from m/z 50 to 1500. The
compounds were detected as m/z [M þ H]þ.
(NEB), to provide a stop for the Taq polymerase downstream
from the primer, and then incubated at pH 7.2 with either com-
pound 5 or brostallicin in the absence or presence of the GSH/
GST system. As a control, plasmid DNA was incubated with the
DNA minor groove binding compound tallimustine at concen-
trations reported to produce alkylation at the 50-TTTTGA target
hexamer (21). After drug treatment, the DNA was precipitated
and washed as described previously (20). The primer was 50-end-
labeled with T4 polynucleotide kinase (NEB) and [γ-32P]ATP
(5000 Ci mmol-1, Amersham). The synthetic primer sequence
and the linear PCR amplification conditions were followed
as described previously (21). Samples were then purified by
extraction with an equal volume of chloroform and isoamyl
alcohol (24:1), precipitated, and washed. Dried samples were
resuspended in loading buffer and denatured at 90 ꢀC for 2 min
before being loaded onto an 8% polyacrylamide denatured gel.
After the run, the gel was dried and autoradiographed.
HPLC-MS Analysis of Brostallicin-GSH Adducts
Formed in Water (no buffer) and in Buffer Solutions. Bro-
stallicin-GSH adduct 6 and a mixture of 6 and 7 were obtained
as reported in Materials. Compounds were controlled using an
Dionex Ultimate 3000 series HPLC system interfaced with a
triple-quadrupole MDS Sciex (Foster City, CA) API 3000 mass
spectrometer equipped with a Turboionspray (ESI). Detection of
positive ions [M þ H]þ was performed in multiple-reaction
monitoring (MRM) mode, monitoring the transition of the m/z
968 precursor ion to the m/z 695 and 839 product ions for
compound 6 and of the m/z 1048 precursor ion to the m/z 645 and
951 product ions for compound 7.
Spectrofluorometric Evidence of the Reaction between
Brostallicin and GSH. GST activity with GSH and brostallicin
as substrates was measured at pH 6.5 in a single-photon counting
spectrofluorometer (Perkin-Elmer LS50B) with a sample holder
set at 25 ꢀC. Excitation was at 370 nm, and emission was collected
at 425 nm. The increase in fluorescence intensity was measured
after the addition of a suitable amount of GST to 1 mL (final
volume) of 0.1 M potassium phosphate buffer (pH 6.5) (adjusted
to pH 6.5 with diluted HCl), containing 0.1 mM EDTA, and
different amounts of substrates. In particular, steady-state reaction
kinetics between GSH and brostallicin were studied with either 1
μM GSTP1-1 or 1 μM GSTM2-2, by varying either the GSH
concentration from 1 to 20 mM (keeping the brostallicin concen-
tration constant at 50 μM) or the brostallicin concentration from
1 to 100 μM (keeping the GSH concentration constant at 1 mM).
The dependence of steady-state velocity on substrate concentra-
tion was fitted to a simple binding isotherm to calculate the
apparent Michaelis constants (Km).
RESULTS
Sensitivity to Brostallicin Is Higher in Cells Overexpres-
sing either the GST-π or the GST-μ Gene. The cytotoxic
activity of brostallicin was evaluated in the human breast
carcinoma MCF-7 parental cell line and in previously character-
ized clones, transfected with the human GST-π, GST-μ, or GST-
R gene, showing GST levels higher than those found in parental
cells (13, 14). GST-π- and GST-μ-overexpressing cells were 9-fold
more susceptible to the cytotoxic activity of brostallicin than the
empty vector-transfected MCF-7 cells (Table 1). Brostallicin was
only 2-fold more cytotoxic in GST-R-transfected MCF-7 cells
which was in agreement with previously published data showing
that the human mu and pi class GST isoenzymes were stronger
activators than the alpha class GST isoenzyme (4). Moreover, the
debrominated derivative of brostallicin, compound 3, when
compared with brostallicin on MCF-7 cells, was found to be
1000-fold less cytotoxic than the parent drug, and its potency was
not dependent of GST expression (data not shown).
HPLC-UV/MS Evidence of the Reaction between
Brostallicin and GSH. The GSH and brostallicin reaction time
course was followed via a HPLC-UV system connected to a
mass spectrometer, permitting compound identification. The rate
of spontaneous reaction between GSH (1 mM) and brostallicin
(100 μM) was negligible at pH 6.5 in the absence of GST.
However, in the presence of either GSTM2-2 (Figure 2A) or
GSTP1-1 (Figure 2B), a decrease in brostallicin concentration
and synchronous formation of a few GSH adducts were ob-
served. Moreover, the reaction in the presence of GSTP1-1
occurred at a rate ∼4 times higher than that with GSTM2-2.
Kinetic Analysis of the Brostallicin-GSH Reaction
Catalyzed by GSTP1-1. The fluorescence time course of the
reaction between brostallicin and GSH in the presence of variable
amounts of GST was fitted to Scheme 1 to yield the microscopic
rate constants and the thermodynamic constants listed in Table 2.
COPASI version 4.4.27 (19) carried out the simultaneous fitting
of the observed time courses to Scheme 1 by means of numerical
integration of the ordinary differential equations. By using
numerical integration, we also obtained the simulated time
dependences of the observed events.
Table 1: Brostallicin Efficacy on a Human Breast Tumor Cell Line
(MCF7) Overexpressing or Not Different GST Isoenzymesa
IC50 (μM) ( SD
drug
MCF-7 neo
MCF-7π
MCF-7μ
MCF-7R
brostallicin
0.55 ( 0.1
0.06 ( 0.00
0.06 ( 0.00
0.24 ( 0.01
Taq Polymerase Stop Assay. This assay is a linear ampli-
fication method employing the properties of DNA polymerase in
investigating the sequence selectivity of the interaction between
DNA-damaging agents and DNA. Studies with the Taq stop
assay were based on a previously reported method (20). Plasmid
pBSSK-TOPO II was linearized with a PstI restriction enzyme
aBrostallicin activity was tested in isogenic human tumor cell lines
differing in the expression of the GST isoenzyme. MCF7 cells overexpres-
sing pi and mu class GST isoenzymes are more sensitive to brostallicin
compared to the parental cells which do not express GST. IC50 is the dose
causing 50% inhibition of colony growth in treated culture vs untreated
controls. Values are means of four independent experiments. SD is the
standard deviation.

Article
Biochemistry, Vol. 49, No. 1, 2010 229
F
IGURE 2: Time course of the brostallicin-GSH reaction followed by the HPLC-UV/MS method. Analysis was performed at different time
intervals on a reaction mixture containing GSH, brostallicin, and either GSTM2-2 (A) or GSTP1-1 (B). For experimental conditions, see
Experimental Procedures. The peak area of each species is expressed as the percent of the brostallicin peak area at time zero. Panels C and D show
the HPLC-UV chromatograms for the reactions with GSTP1-1 (at 10 min) and GSTM2-2 (at 30 min), respectively. Panels E-G show the ESI
mass spectra of compounds 6, 7, and 5. Under the experimental conditions utilized, both the singly and doubly charged peaks were detected for
each compound. The inset of panel G is an expanded view of the peak at m/z 986 (compound 5) showing the characteristic splitting due to the
presence of two isotopes of chlorine, 35Cl and 37Cl.

230 Biochemistry, Vol. 49, No. 1, 2010
Pezzola et al.
F
IGURE 3: Inhibition of GSTP1-1 and GSTM2-2 by either brostallicin or compound 3, 6, or 7. Experiments were performed at pH 6.5 and
25 ꢀC. (A-D) The brostallicin concentration was varied between 0 and 50 μM with GSTP1-1 and between 0 and 1 μM with GSTM2-2.
At each inhibitor concentration, the concentration of either GSH or CDNB was varied from 0.25 to 2 mM, while the cosubstrate concen-
tration was maintained constant at 1 mM. Initial velocity data are reported as Lineweaver-Burk plots. (E) The concentration of the debro-
minated derivative of brostallicin, compound 3, was varied between 0 and 50 μM, while the concentrations of CDNB and GSH were main-
tained constant at 1 mM. (F) The concentrations of compounds 6 and 7 were varied between 0 and 20 μM, while the concentrations of CDNB
and GSH were maintained constant at 1 mM: compound 6 (0) and a mixture of compounds 6 and 7 (9). Data are reported as the percent of
GST inhibition, and the solid lines represent the best fit of data to a simple binding isotherm. Points are means of four independent experiments;
bars show the standard deviation. (G and H) HPLC-MS analysis of compound 6 (G) and a mixture of compounds 6 and 7 (H) obtained
enzymatically as reported in Experimental Procedures. The total ion chromatograms of the MRM transitions of compounds 6 (m/z 968/695
and 968/839) and 7 (m/z 1048/645 and 1048/951) are shown. The chromatograms are acquired by simultaneously selecting the MRM transitions
of both compounds 6 and 7.

Article
Biochemistry, Vol. 49, No. 1, 2010 231
converted into products within the experimental observation
time. The initial velocity data obtained from inhibition studies are
represented as Lineweaver-Burk plots. The patterns obtained
suggest that brostallicin behaves like a competitive inhibitor of
GSTP1-1, with respect to the cosubstrate CDNB (Figure 3B),
while brostallicin behaves more like an uncompetitive inhibitor
with respect to GSH (Figure 3A). With GSTM2-2, brostallicin
behaves like a mixed-type inhibitor with respect to both sub-
strates (Figure 3C,D). Inhibitor constants were determined by
replot analysis of the primary reciprocal plots according to the
method of Segel (18). With GSTP1-1, the inhibition constants are
as follows: KiGSH = 28 ( 0.1 μM, and KiCDNB = 37 ( 0.1 μM.
With GSTM2-2, the mixed-type inhibition yields two series of Ki
values: Ki values for the competitive component (KiGSH = 0.6 (
0.1 μM, and KiCDNB = 0.4 ( 0.1 μM) and Ki values for the
uncompetitive component (KiGSH = 1.0 ( 0.4 μM, and KiCDNB
=
1.7 ( 0.3 μM). Therefore, brostallicin is much more efficient in
inhibiting GSTM2-2 than in inhibiting GSTP1-1.
Bromine is an essential structural requirement for a tight
interaction with the GST isoenzymes. In fact, the debrominated
derivative of brostallicin (compound 3) is almost completely
ineffective as an inhibitor of GSTP1-1 and shows, with GSTM2-
2, an IC50 of approximately 60 μM, a value 100 times higher than
that observed with brostallicin under the same experimental
conditions (GSH and CDNB concentrations constant at 1
mM) (Figure 3E).
F
IGURE 4: Brostallicin-GSH adducts 6 and 7 do not cause irrever-
sible inhibition of GSTs. GSTM2-2 (A) and GSTP1-1 (B) were
incubated at a constant GSH (1 mM) and brostallicin concentrations
(100 μM with GSTM2-2 and 400 μM with GSTP1-1). After a suitable
incubation time for conversion of brostallicin in its GSH derivatives,
different aliquots of the incubation mixture were assayed for GST
activity after dilution in 1 mL (final volume) of standard assay
mixture. All experiments were performed in triplicate. Data are
expressed as means ( the standard deviation.
We also analyzed the inhibition effect of GSH-brostallicin
adducts 6 and 7 on the reaction between CDNB and GSH
catalyzed by GSTP1-1. When a mixture of these compounds was
utilized, a quite hyperbolic inhibition trend was obtained
(Figure 3F) in which one phase was clearly resolved (IC50 ∼ 1
μM). An identical behavior was observed when the experiments
were repeated employing only compound 6 (Figure 3F). Com-
pounds 6 and 7 differ only in the group bound to the R-carbon of
the carboxamido moiety. Therefore, we assume that this group
does not play a role in the interaction with GSTP1-1 and that the
affinity of both compounds 6 and 7 for the enzyme is similar to
that of the transient R-chloroamido species 5.
Brostallicin and Its GSH Adducts Do Not Irreversibly
Inactivate GSTP1-1 and GSTM2-2. The interaction of GSTs
with R,β-unsaturated compounds, such as brostallicin, typically
leads to irreversible inhibition of these enzymes (25). The same
mechanism can be hypothesized for reactive intermediates 4 and
5, which might covalently interact with the nucleophilic residues
of the enzyme. To evaluate the possibility of a covalent inhibition,
we subjected either GSTP1-1 or GSTM2-2 to a prolonged
incubation with brostallicin and GSH to convert brostallicin
quantitatively into its GSH derivatives. Then, we determined the
effect of dilution on the degree of enzyme inhibition. Complete
restoration of the original catalytic activity was observed after
dilution, ruling out the possibility of an irreversible inactivation
of the GSTs (Figure 4).
The main product of both reactions was represented by a species
at m/z 968, identified as brostallicin-GSH adduct 6 in which
bromine is replaced by one hydroxy group (Figure 2C-E).
Another product at m/z 1048 was identified as brostallicin-GSH
adduct 7 in which bromine is replaced by one phosphate group
(Figure 2C,D,F). GSH-brostallicin R-bromoamido derivative 4
was not detected under these conditions, confirming the hypoth-
esis that brostallicin, after the addition of GSH, forms a highly
reactive intermediate which substitutes bromine with other
species present in solution (22-24). However, a long-lived
transient species has been observed at m/z 986. This intermediate
has been identified as GSH-brostallicin R-chloroamido deri-
vative 5 in which bromine is replaced by chlorine ions from the
reaction medium (Figure 2C,D,G). The presence of chlorine
in this species is confirmed by the mass spectrum related to the
corresponding chromatographic peak where the protonated
molecular ion shows the characteristic splitting and abundance
ratio of two isotopic species, 35Cl and 37Cl (Figure 2G, inset).
During the experiment, this transient reactive species under-
goes a relatively slow (t1/2 ∼ 45-50 s) conversion to stable
R-hydroxy derivative 6 and R-phosphate derivative 7. This
hypothesis is further supported by the evidence that when the
reaction medium was deprived of chloride ions, using sodium
acetate buffer, the transient species at m/z 986 was not observed
while a new acetate derivative (compound 8) was detected (data
not shown).
Spectrofluorometric Evidence of the Reaction between
Brostallicin and GSH. The low intrinsic fluorescence of
brostallicin undergoes a time-dependent increase in the presence
of GSH and either GSTP1-1 or GSTM2-2 (Figure 5A). This
spectral change of brostallicin does not occur in the absence of
either GSH or GST. Thus, the increase in the fluorescence of
brostallicin has been attributed to the exchange of the halogen
group with either the hydroxy or the phosphate group present in
solution, giving compound 6 or 7, respectively. The rate of this
event increases linearly with the amount of GST (Figure 5B),
Inhibition of GSTP1-1 and GSTM2-2 Isoenzymes by
Brostallicin and Its Derivatives. The affinity of brostallicin for
human isoenzymes GSTP1-1 and GSTM2-2 was estimated at pH
6.5 by following the effect of increasing drug concentrations on
the initial velocity of the reaction between GSH and CDNB.
Since the rate of reaction between GSH and CDNB is more than
100 times higher than the rate of reaction between GSH
and brostallicin, we can assume that almost no brostallicin is

232 Biochemistry, Vol. 49, No. 1, 2010
Pezzola et al.
F
IGURE 5: Spectrofluorometric evidence of the reaction between GSH and brostallicin. (A) The fluorescence spectra of 50 μM brostallicin in 0.1
M potassium phosphate buffer (pH 6.5) were recorded in the absence ( ) and presence of 1 mM GSH (---), 1 mM GSH, and 0.2 μM GSTP1-1
3 3 3
(;). Spectra were recorded at different incubation times from 1 to 40 min (excitation at 370 nm). (B) Rate of spectral change (V) as function of
GST concentration. Brostallicin (25 μM) was reacted at pH 6.5 and 25 ꢀC with GSH (1 mM) in the presence of an increasing amount of GSTP1-1
(from 0.5 to 4 μM). The reaction was followed at 425 nm (excitation at 370 nm) by fluorometry, and the velocity was measured immediately after
the addition of the enzyme. (C-F) Effect of either brostallicin or GSH concentration on the initial velocity of the reaction. Experiments were
conducted at pH 6.5 and 25 ꢀC, with either 1 μM GSTM2-2 (C and E) or 1 μM GSTP1-1 (D and F). We obtained the dependence of the initial
velocity on GSH concentration by varying the GSH concentration from 1 to 20 mM and keeping the brostallicin concentration constant at 50 μM
(C and D). We analyzed the effect of brostallicin by varying the brostallicin concentration from 1 to 100 μM and keeping the GSH concentration
constant at 1 mM (E and F). The solid lines represent the best fit of data to a simple binding isotherm. The experimental data reported in panel F
were also fitted to a sigmoidal equation ( ) to highlight the nonhyperbolic trend. The conversion of arbitrary fluorescence units into nanomoles
of product was based on a calibration curve built using known amounts of preformed compounds 6 and 7. All experiments were performed six
times. Data are expressed as means ( the standard deviation.
3 3 3
confirming that the enzyme promotes the reaction leading to
increased fluorescence. On the basis of this evidence, we have
developed a new continuous fluorometric assay for studying the
kinetics of the reaction between brostallicin and GSH in the
presence of GSTs. Steady-state kinetic analysis of the reaction
confirms that GSTM2-2 (kcat/Kmbrostallicin = 4 mM-1 s-1) is a
less efficient catalyst toward brostallicin compared to GSTP1-1
(kcat/Kmbrostallicin = 11 mM-1 s-1). The specificity constants
reported above were calculated from initial velocity versus
brostallicin concentration plots, obtained at saturating GSH
concentrations and after subtraction of the rate of the none-
nzymatic reaction (data not shown). Moreover, in the presence of
GSTM2-2, the apparent Michaelis constants for GSH and
brostallicin (KmGSH = 4.5 mM, at 50 μM brostallicin, and
Kmbrostallicin = 5 μM, at 1 mM GSH) are higher than the apparent
dissociation constants found for these substrates [KdGSH e 50
μM (26), and Kibrostallicin ∼ 0.5 μM] (Figure 5C,E). This suggests
that GSTM2-2 does not follow a rapid equilibrium Michae-
lis-Menten mechanism. However, the low catalytic activity of
the GSTM2-2 isoenzyme and its high affinity for brostallicin
prevent an accurate kinetic study.
The dependence of GSTP1-1 activity on brostallicin concen-
tration shows a cooperative trend (Figure 5F); therefore, a
more detailed analysis of the reaction between GSH and bros-

Article
Biochemistry, Vol. 49, No. 1, 2010 233
Scheme 1: Kinetic Scheme for the Reaction Catalyzed by
GSTP1-1a
Evidence at Physiological pH: Brostallicin Activation
Requires GST, and the R-Chloro Derivative 5 Binds Cova-
lently to DNA. The reactivity of nucleophiles is influenced by
pH; therefore, the reaction between brostallicin and GSH might
be enzyme-independent at physiological pH. However, we ob-
served that 10 μM brostallicin [the highest drug concentration
utilized on tumor cells (7)] reacts very slowly with GSH at pH 7.4
and GSTs are required to achieve an effective brostallicin
activation rate (Figure 6A). Moreover, we tested the ability of
either brostallicin or its R-chloro derivative 5 to interact with
DNA at physiological pH. In fact, DNA minor groove binders
make up a class of anticancer agents whose activity in tumor cells
has been correlated to their ability to alkylate DNA in a highly
sequence-specific manner (27). The sequence-specific covalent
DNA interaction of brostallicin was analyzed by the Taq
polymerase stop assay. The polyacrylamide gel autoradiography
reported in Figure 6B shows that brostallicin alone, in the ab-
sence of the GSH/GST system, did not alkylate DNA (lane 5),
while a band was observed when the GSH/GST system was
added to the reaction mixture (lane 6). Compound 5 in the
absence of was GSH/GST system able to bind covalently to
DNA (lane 7) and with the same sequence specificity as bros-
tallicin in the presence of the GSH/GST system. Both brostallicin
and compound 5 bind to a sequence (AAAG) that differs from
the DNA minor groove binding specificity of tallimustine (lanes
3 and 4). Thus, our data indicate that the GSH/GST system
converts brostallicin into the reactive molecule 5 which is able to
covalently interact with the same DNA sequence of brostallicin.
These data support the hypothesis that compound 5 may
represent the critical intermediate in the activation mechanism
of brostallicin (Scheme 2).
aThe mechanism assumes that the enzyme consists of two sub-
units that are in identical conformations and behave indepen-
dently; thus, only one subunit is shown. Moreover, GSH
(present at a high and almost constant concentration) is omitted
for the sake of simplicity. The catalytic step involves the rapid
formation of the intermediate product (P) and its slow conver-
sion into the final fluorescent product(s) (F). Abbreviations: E,
GSTP1-1; B, brostallicin; P, compound 5; F, mixture of com-
pounds 6 and 7.
Table 2: Kinetic Parameters for the Brostallicin-GSH Reaction Catalyzed
by GSTP1-1a
kinetic parameter
value ( SD
BKd (μM)
50 ( 19
0.6 ( 0.1
3 ( 1
k1 (s-1
)
PKd (μM)
k2 (s-1
)
0.026 ( 0.001
aKinetic and thermodynamic constants were obtained by the best fit to
Scheme 1 of the fluorescence traces of the reaction between GSH and
brostallicin at different GSTP1-1 concentrations. BKd is the dissociation
constant for the binding of brostallicin to GSTP1-1. k1 is the rate constant
for the conversion of brostallicin into the transient product P. PKd is the
dissociation constant for the binding of P to GSTP1-1. k2 is the rate
constant for the conversion of P into the fluorescence product(s) (F). SD is
the standard deviation.
DISCUSSION
Since 2001, brostallicin has been extensively investigated,
because of its promising antitumor activity profile in experi-
mental models and its enhanced activity in cancer cells with high
GSH/GST levels (7). This distinctive activation mechanism
exploits a recognized vulnerability found in many cancer cells,
as the GSH/GST detoxifying pathways are often upregulated in
human tumors and a cause of reduced responsiveness (i.e.,
chemoresistance) to commonly used anticancer agents. However,
direct evidence of the interaction between brostallicin and the
GSH/GST system and the mechanism of drug activation was
lacking. In this work, we have provided evidence that the
GSH/GST system plays a key role in determining the sensitivity
of cells, as overexpression of mu and pi class GSTs increases
brostallicin cytotoxicity. We clearly show that brostallicin binds
both GSTP1-1 and GSTM2-2, the most abundant GST iso-
enzymes in tumor cells, with an affinity for GSTM2-2 almost
2 orders of magnitude higher than that for GSTP1-1. A careful
investigation of the H-site’s architecture of these isoenzymes
reveals that residues Ile104 and Arg13, in the GSTP1-1 active site,
are replaced with Ala111 and Leu12 in GSTM2-2 (29, 30).
Therefore, the cumbersome brostallicin molecule may better
accommodate the large active site cleft of the mu class isoenzyme.
Both GST isoenzymes catalyze the addition of GSH to brostalli-
cin. GSTM2-2 catalyzes the reaction at a rate lower than that of
GSTP1-1; however, given its high affinity, it is fully active at
brostallicin concentrations that are effective on tumor cells. This
suggests that brostallicin may also be suitable for the treatment of
tumors selectively expressing mu class GST. A minimum reaction
tallicin catalyzed by GSTP1-1 has been performed to define this
behavior.
Kinetic Analysis of the Brostallicin-GSH Reaction
Catalyzed by GSTP1-1. We carefully analyzed the fluores-
cence time courses of the reaction of GSH and brostallicin in the
presence of different amounts of GSTP1-1. The experimental
traces were best fitted to a minimal reaction mechanism
(Scheme 1), to yield the microscopic rate constants and the
thermodynamic constants listed in Table 2. In the proposed
kinetic scheme, the catalytic step involves the rapid formation of
a GSH-brostallicin intermediate (P), identified as compound 5,
which slowly converts into the final fluorescent product(s) (F),
identified as a mixture of compounds 6 and 7. The presence of this
second slow reaction explains well both the autocatalytic fluor-
escence traces (data not shown) and the nonhyperbolic depen-
dence of the initial velocity on brostallicin concentration
(Figure 5F). The estimation of the thermodynamic constants
utilized for fitting the experimental data to Scheme 1 has been
derived from different experimental approaches. (a) The disso-
ciation constant for the binding of brostallicin to GSTP1-1 was
obtained by inhibition studies (see Figure 3A,B) using brostalli-
cin as an inhibitor of the reaction between CDNB and GSH. (b)
The dissociation constant for the binding of the product(s) to
GSTP1-1 was obtained by inhibition experiments using com-
pounds 6 and 7 as inhibitors of the reaction between CDNB and
GSH (Figure 3F).

234 Biochemistry, Vol. 49, No. 1, 2010
Pezzola et al.
F
IGURE 6: (A) Fluorescence time courses of the reaction between GSH (1 mM) and brostallicin (10 μM), performed at pH 7.4 and 25 ꢀC in the
absence (spontaneous) and presence of either GSTP1-1 (1 μM) or GSTM2-2 (1 μM). (B) Autoradiography of a typical Taq stop assay on
topoisomerase II cDNA after drug treatment. Compound 5 was able to bind covalently to DNA in the absence of the GSH/GST system (lane 7).
Brostallicin alone did not alkylate DNA (lane 5), while a band was observed when the GSH/GST system was added to the reaction mixture (lane
6). In the absence of drugs, the GSH/GST system did not induce any alteration able to block Taq polymerase (lanes 1 and 2). As a control,
tallimustine was tested (lanes 3 and 4). The experiment was performed as described in Experimental Procedures. Arrows denote the alkylated
regions.
Scheme 2: Proposed Mechanism for Brostallicin Activation by the GST/GSH System (8, 28)
mechanism has been proposed to satisfactorily describe the
GSTP1-1-catalyzed reaction of GSH with brostallicin. This
mechanism implies that the enzyme catalyzes the formation of
one intermediate brostallicin derivative which is slowly converted
into a few stable products. This agree with HPLC-UV/MS
analysis which reveals the presence of a transient species
(compound 5) that slowly reacts with the surrounding species
generating fluorescent derivatives 6 and 7. These compounds are
very stable in solution as the R-hydroxy (compound 6) and
R-phosphate (compound 7) are bad leaving groups. Compound 5
could be inactivated by substitution of the reactive chlorine with
an additional GSH molecule whose concentration is in the
millimolar range (0.5-10 mM) in mammalian cells. However,
under our experimental conditions, the amount of derivative
bearing a second molecule of GSH is negligible; this may be due
to the steric hindrance between the two GSH molecules. The
relatively long living intermediate 5 (t1/2 ∼ 45-50 s under our
experimental conditions) is very likely transiently populated to a
significant amount also inside the cells, where chloride ions are
abundant, and may represent the real active form of the drug.
This hypothesis is supported by the Taq polymerase stop assay
showing that compound 5 is able to bind covalently to DNA with
the same DNA sequence specificity as the parent drug, in the
presence of both GSH and GSTs.
The mechanism of the reaction herein described for brostalli-
cin is similar to that reported by Creighton et al. (31) for the
activation of 2-crotonyloxymethyl-2-cycloalkenone drugs. Like-
wise, a substrate is transformed into a toxic product by GST. The
reaction is a multistep process, involving the formation of a
reactive GS conjugate that reacts spontaneously with other GSH
molecules giving stable glutathionylated adducts. The apparent
rates of formation and degradation of the active species are on the
same order of magnitude as the rates we observed, leading to the
potential therapeutic use of these pro-drugs. In conclusion, we
have explained why, unlike that of most anticancer drugs (32),
brostallicin efficacy takes advantage of high intracellular levels of

Article
Biochemistry, Vol. 49, No. 1, 2010 235
both GSH and GSTs. Our data both reinforce and further the
understanding of the targeted activation of brostallicin. Con-
sidering that several human tumors display increased levels of pi
and/or mu class GSTs (12, 13), these data highlight how this
activation relates to the established anticancer activity of bros-
tallicin.
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17. Gill, S. C., and von Hippel, P. H. (1989) Calculation of protein
extinction coefficients from amino acid sequence data. Anal. Biochem.
182, 319–326.
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