com
JOURNAL OF
Inorganic
Biochemistry
Journal of Inorganic Biochemistry 102 (2008) 564575
www.elsevier.com/locate/jinorgbio
Received 2 July 2007; received in revised form 1 October 2007; accepted 18 October 2007
Available online 28 November 2007
Abstract
Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents.
During the past decade a number of structurally diverse gold(III) complexes were reported to be acceptably stable under physiological-
like conditions and to manifest very promising cytotoxic eects against selected human tumour cell lines, making them good candidates
as anti-tumour drugs. Some representative examples will be described in detail. There is considerable interest in understanding the precise
biochemical mechanisms of these novel cytotoxic agents. Based on experimental evidence collected so far we hypothesize that these
metallodrugs, at variance with classical platinum(II) drugs, produce in most cases their growth inhibition eects through a variety of
DNA-independent mechanisms. Notably, strong inhibition of the selenoenzyme thioredoxin reductase and associated disregulation
of mitochondrial functions were clearly documented in some selected cases, thus providing a solid biochemical basis for the pronounced
proapoptotic eects. These observations led us to investigate in detail the reactions of gold(III) compounds with a few model proteins in
order to gain molecular-level information on the possible interaction modes with possible protein targets. Valuable insight on the for-
mation and the nature of goldprotein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectropho-
tometric studies of appropriate model systems as it is exemplied here by the reactions of two representative gold(III) compounds with
cytochrome c and ubiquitin. The mechanistic relevance of gold(III)-induced oxidative protein damage and of direct gold coordination to
protein sidechains is specically assessed. Perspectives for the future of this topics are briey outlined.
2007 Elsevier Inc. All rights reserved.
Keywords: Gold(III) complexes; Anticancer agents; Proteins; ESI mass spectrometry; Mechanism of action
1. Novel gold(III) compounds as anticancer agents: pharmacological prole of cisplatin suggested that other
structurally diverse compounds with outstanding metal-based compounds might pairwise manifest impor-
antiproliferative properties tant anti-tumour eects while (ideally) exhibiting a dierent
spectrum of biological activities and a lower systemic tox-
The discovery of the anticancer properties of cisplatin icity [2,3]. As d8 Au(III) complexes are isoelectronic and
during the 1960s triggered a great deal of interest in the isostructural with Pt(II) complexes, square planar gold(III)
eld of anti-tumour metallodrugs [1]. The very favourable compounds soon appeared to be excellent candidates for
anticancer evaluation. However, at variance with plati-
*
Corresponding authors. Fax: +39 055 457 3385.
num(II) compounds, gold(III) analogues were found to
E-mail addresses: angela.casini@uni.it (A. Casini), luigi.messori@ manifest, on the whole, a rather poor stability prole being
uni.it (L. Messori). kinetically more labile than the corresponding platinum(II)
0162-0134/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2007.11.003
A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 565
compounds, light-sensitive and easily reducible to metallic The antiproliferative properties of these gold(III)poly-
gold. As a result of these diculties and, also, of detection amine were, then, measured by the sulforhodamine B assay
of important systemic toxicity in the course of the rst ani- on the representative human ovarian tumour cell line
mal studies, gold(III) compounds were quickly abandoned. A2780, either sensitive (A2780/S) or resistant (A2780/R)
Nonetheless, during the 1990s, renewed interest for to cisplatin. In most cases, the above compounds revealed
anticancer gold(III)-based compounds emerged place, a high cytotoxicity, with IC50 values generally falling in the
especially when a few novel gold(III) complexes, exhibit- low micromolar region [9], and were also found to over-
ing improved stability, lower toxicity and favourable come resistance to cisplatin in the cisplatin-resistant line.
in vitro pharmacological properties, were made available Later on, in collaboration with the group of Minghetti
for pharmacological testing [4]. For instance, a series of and Cinellu, we characterised and assayed other gold(III)
organogold(III) DAMP compounds (DAMP = 2-[(dimeth- complexes, bearing the bipyridyl motif [10]. This family of
ylamino)methyl]phenyl) were developed by Buckley et al., compounds turned out to display appreciable stability in
and screened for anti-tumour activity with encouraging solution and to cause relevant tumour growth inhibition
results in vitro [5,6]. Further these compounds demon- in vitro. In particular, our investigations focused on two
strated moderate activity in laboratory animals [5,6]. members of this family, namely [Au(bipyc-H)(OH)][PF6] 6
Later on, in the attempt of nding new biologically (where bipyc = 6-(1,1-dimethylbenzyl)-2,20 -bipyridine) and
active substances with an even better stability prole, some [Au(bipy)(OH)2][PF6] 7 (bipy = bipyridine) (Fig. 2) [9]. A
classical square planar gold(III) complexes, based on a number of analogues were also prepared and characterised
variety of structurally dierent ligands, were prepared (see Fig. 2, compounds 8 and 9) [11].
and characterised in our laboratory [7]. To improve the sta- In [Au(bipy)(OH)2][PF6] 7, the gold(III) center is coordi-
bility of the gold(III) center, polydentate ligands such as nated by two nitrogens of the bidentate bipyridyl ligand and
polyamines, cyclam, terpyridine and phenathroline were by two hydroxide groups. At variance, 6 is an organo-
preferentially employed. A few compounds, namely gold(III) complex in which donors to the gold(III) center
[Au(en)2]Cl3 1, [Au(dien)Cl]Cl2 2, [Au(cyclam)](ClO4)2Cl are two nitrogens from the bipyridyl moiety, the C2 carbon
3, [Au(terpy)Cl]Cl2 4, and [Au(phen)Cl2]Cl 5, (Fig. 1), were of the phenyl group, and a hydroxide group. Only small
characterised both in the solid state and in solution [8]. On deviations from ideal square planar geometry were seen in
the whole, a quite satisfactory stability prole in solution the classical bipyridyl complexes whereas such deviations
emerged for all these gold(III) compounds that opened are quite large in the case of cyclometallated derivatives.
the way to their in vitro pharmacological testing. This Both mentioned compounds exhibited sucient solubil-
means that the donor set comprising three or four nitrogen ity in watery solutions. Their intense LMCT visible bands,
atoms, produces a strong stabilisation of the gold(III) cen- lying in the 300370 nm region and diagnostic of gold in
ter and a net decrease of the reduction potential, thus pre- the oxidation state +3, were exploited to monitor reactions
venting gold(III) reduction and hydrolysis. with various kinds of biomolecular targets. Notably, these
3+
2+
NH2 3+
NH2
NH
NH NH 2ClO4-
Au
3Cl- Au Au Cl-
NH2 NH2
NH2 NH2
Cl
NH NH
1 2
3
2+
N N
Cl
N Au Cl Cl
2Cl Au
N N Cl
4 5
Fig. 1. Schematic drawing of (1) [Au(en)2]Cl3, (2) [Au(dien)Cl]Cl2, (3) [Au(cyclam)](ClO4)2Cl, (4) [Au(terpy)Cl]Cl2, and (5) [Au(phen)Cl2]Cl.
566 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
+ +
In the same period, Fregona and coworkers prepared
and characterised some interesting gold(III) dithiocarba-
N N CH3 PF6- N N PF6- mate compounds displaying a very encouraging biological
Au Au prole [14]. The compounds containing the N,N-di-
OH CH3
OH OH methyldithiocarbamate and ethylsarcosinedithiocarbamate
ligands (compounds 10 and 11 in Fig. 2) were the most
7
6 intensely studied ones. Early in vivo data on murine models
looked very promising and led to the rapid patenting of
+
these novel compounds. Afterwards, some additional
N N H3COOC N investigations were carried out aimed at elucidating the
CH3 CH3
Au PF6- Au main features of their mechanism of action, both at the cel-
CH3 HN CH3 H3COOC CH3 lular and biochemical level [15,16]. It is worth noting that
very recent results suggest that these gold(III) dithiocarba-
CH3 mate compounds most likely act through inhibition of the
9
8
O cancer cell proteasome [17], a quite novel and unexpected
H3C S X S X
mechanism for anticancer metallodrugs.
C
N C Au Au
Concomitantly, a series of interesting gold(III) meso-tet-
RO CH2 N C
raarylporphyrins complexes were developed by Chi Ming
H3C S X CH3 S X
X = Cl, Br Che, and coworkers at the University of Hong Kong [18].
10 R = CH3CH2 11 These compounds, of general formula [Au(III)(p-Y-
TPP)]Cl, [with Y being H (a), Me (b), OMe (c), Br (d) or
Fig. 2. Schematic drawing of [Au(bipyc-H)(OH)][PF6] (6), [Au(bipy)
Cl (e)] (see Fig. 3), were characterised by classical physio-
(OH)2][PF6] (7), [Au(bipydmb-H)(2.6-xylidine-H)][PF6] (8), [Au(pydmb-H)-
(AcO)2] (9) (where bipydmb = 6-(1,1-dimethylbenzyl)-2,20 -bipyridine); chemical methods [18]. Interestingly, coordination to the
pydmb = 2-(1,1-dimethylbenzyl)-pyridine)) and of the gold(III) dithiocar- tetrapyrrole ring causes a large stabilisation of the gold(III)
bamate complexes containing N,N-dimethyldithiocarbamate (10) and oxidation state leading to its high stability in aqueous solu-
ethylsarcosinedithiocarbamate (11) ligands. tions even in the presence of glutathione (GSH), the most
important intracellular reducing agent.
two complexes revealed a clearly dierent reactivity toward All the mentioned gold(III) porphyrins displayed excel-
ascorbate as only compound 7 was shown to be reduced. lent in vitro antiproliferative eects, with IC50 values of
This nding implies that the oxidation state +3 is far more 0.11.5 lM. The lack of cross-resistance with classical
stable in the case of the organogold(III) species compared platinum(II) compounds again suggests that gold(III) por-
to compound 7, in agreement with electrochemical results phyrins and cisplatin induce cytotoxicity through quite dis-
[12]. tinct mechanisms. Remarkably, a zinc(II) analogue [Zn(II)
The in vitro cytotoxic properties of these bipyridyl (TPP)] was also investigated and found to be at least 100-
Au(III) complexes were measured toward the human ovar- fold less cytotoxic than gold(III) porphyrins (IC50 >
ian carcinoma cell line A2780, either sensitive or resistant
to cisplatin. Both gold(III) complexes showed excellent
cytotoxic eects, with IC50 values staying in the low micro-
Y
molar range [9,10]. Compound 6 turned out to be the most
active with a twofold higher activity than cisplatin in the
A2780/R cell line. The antiproliferative properties of these
complexes were also investigated on the human ovarian cell
line SKOV3 (inherently resistant to cisplatin) and on the
+
CCRF-CEM leukemic cell line, either sensitive (CCRF-
CEM/S) or resistant (CCRF-CEM/R) to cisplatin: signi-
N N
cantly lower activities were measured on all these latter
Y Au Y
lines. In any case, an appreciable activity was retained
N N
toward the cisplatin-resistant A2780/R and CCRF-CEM/
R lines suggesting that the biochemical mechanisms of
resistance to cisplatin most likely a more ecient intracel-
lular detoxication and an increased repair of DNA dam-
age are modestly eective toward these gold(III)
complexes. In addition, independent studies pointed out
quite unambiguously that the interactions of these com-
plexes with nucleic acids are weak whereas stronger Y
adducts are formed in the reactions with model proteins Fig. 3. Gold(III) meso-tetraarylporphyrins complexes. Y = H (a), Me (b),
and serum proteins [13]. OMe (c), Br (d), Cl (e).
A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 567
50 lM), thus stressing the crucial role of the gold(III) in vitro antiproliferative properties measured against the
center in the biological action. Nonetheless, the porphyrin reference A2780 human ovarian carcinoma cell line [21].
ligand was found to be essential for anticancer activity While ve compounds of this series manifested only
leading these authors to conclude that the porphyrin ligand moderate cytotoxic properties (with IC50 typically falling
is crucial both in stabilising the Au(III) center and in deliv- in the 1030 lM), the sixth one (Auoxo6), turned out to
ering the metal to its cellular targets [18]. be 515 times more active against both cell lines, thus
A quite common strategy in the eld of anticancer meriting further investigations. In particular, much atten-
metallodrugs is the design and synthesis of polymetallic tion was focused on the chemical and structural reasons
compounds, derived from the fusion of two or more for the higher biological activity of Auoxo6. Remarkably,
monometallic molecular fragments. It follows that the spe- a rather evident positive correlation was identied in a sub-
cic reactivity of each metal center in the resulting com- sequent study between the oxidising power of this com-
pound is further controlled by its interactions with the pound, its reactivity with biomolecular targets and its
nearby metal center(s) and by the overall molecular scaf- antiproliferative eects.
fold. Incorporation of two (or more) metal centers within In Table 1 we have summarised the cytotoxicity data for
an extended molecular framework may greatly aect the the above mentioned gold(III) compounds developed in
overall charge of the resulting polynuclear compound, its our group. According to this data the most active com-
redox properties, the kinetics of hydrolysis, and its specic pounds, compared to cisplatin, are compound 4 and
reactivity toward biomolecules in comparison to mononu- Auoxo6. For these two complexes the extension of the bio-
clear analogues. Some interesting examples of this strategy logical screening over a wider series of cancer cell lines is
are available for platinum(II) and ruthenium(III) antican- now in progress in our group. Furthermore, these most
cer metallodrugs [19,20]. promising molecular scaolds will be used for the drug
These observations prompted us to prepare novel dinu- design of novel gold(III) complexes. For example we have
clear gold(III) species to be tested as anticancer agents, recently synthesised the gold(III)-dinuclear derivative of 4
starting from the above described mononuclear gold(III) whose cytotoxicity properties are under evaluation.
bipyridyl complexes. A series of six dinuclear oxo gold(III) Overall, in the course of the last decade, thanks to the
complexes with bipyridyl ligands (Fig. 4), of general for- eorts of a few research laboratories a signicant number
mula [Au2(N,N)2(l-O)2][PF6]2 [where N,N = 2,20 -bipyri- of structurally diverse gold(III) compounds were prepared
dine (Auoxo1), 4,40 -di-tert-butyl- (Auoxo2), 6-methyl- and characterised that revealed attracting antiproliferative
(Auoxo3), 6-neopentyl- (Auoxo4), 6-(2,6-dimethylphenyl)- activities. In view of the great structural variety of the
(Auoxo5), 6,60 -dimethyl-2,20 -bipyridine (Auoxo6)] was thus investigated gold(III) species it is reasonable to assume that
prepared and characterised in our laboratory, and their their biological eects may depend on dierent biochemical
Me 3C CMe3
N O N N O N
Au Au [PF6]2 Au Au [PF6]2
N O N N O N
Me 3C CMe3
Auoxo1 Auoxo2
R Me Me
N O N N O N
Au Au [PF6]2 Au Au [PF6]2
N O N N O N
R Me Me
Auoxo3: R = Me Auoxo6
Auoxo4: R = CH2CMe 3
Auoxo5: R = C6H3Me2-2,6
Fig. 4. Schematic drawings of the dinuclear gold(III) complexes Auoxo. Auoxo3 is a ca. 1:1 mixture of the cis and trans isomer while Auoxo4 and Auoxo5
are, as depicted, exclusively trans isomers.
568 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
Table 1
Cytotoxicity (IC50-lM) of the gold compounds studied in Florence during the last years towards dierent tumour cell lines
Compounds A2780/S A2780/R CCRF-CEM/S CCRF-CEM/R SK-OV-3 MCF7 HT29 A549
Cisplatin 1.2 0.43 14 2.72 0.7 0.1 20.1 7.2 5.2 5.30 0.87 6.30 0.23
1 [Au(en)2]Cl3 8.36 0.77 17.0 4.24
2 [Au(dien)Cl]Cl2 8.2 0.93 18.7 2.16 12.6 2.0 32.7 6.6
3 [Au(cyclam)]ClO4)2Cl 99.0 >120.0
4 [Au(Terpy)Cl]Cl2 0.2 0.37 0.032
5 [Au(Phen)Cl2]Cl 3.8 1.1 3.49 0.91 2.3 6
6 [Au(bipyc-H)(OH)][PF6] 3.3 1.4 8.2 1.5 11.9 2.1 51.2 5.6 13.3 1.6 35.30 8.8 24.60 >50
7 [Au(bipy)(OH)2][PF6] 8.8 3.9 24.1 8.7 52.9 11.6 58.6 0.9 34.4 4.7
8 [Au(bipydmb-H)(2.6-xylidine- 2.50 0.43 5.7 0.3 5.20 0.40 25 35
H)][PF6]
9 [Au(pydmb-H)(AcO)2] 2.90 0.34 6.40 1.0 7.70 0.44 8.60 49
Auoxo1 22.8 1.53 23.3 0.35
Auoxo2 12.1 1.5 13.5 1.8
Auoxo3 25.4 2.47 29.8 3.1
Auoxo4 12.7 1.06 19.8 1.8
Auoxo5 11.0 1.5 13.2 1.2
Auoxo6 1.79 0.17 4.81 0.5
Cisplatin is reported as reference compound. Data were collected after 72 h exposure to drug.
mechanisms. Current eorts to identify those mechanisms analysed at the molecular level by taking advantage of a
are described in the next section. In any case sucient vast array of biochemical, biophysical and physicochemical
evidence has been collected so far that the presence of a methods [13,22].
gold(III) species is usually associated with outstanding A representative example is the case of gold(III) porphy-
cytotoxic properties so that the metal itself seems to be rins whose reactions with nucleic acids were investigated.
one of the major determinants of the biological actions. In particular, Sun and coworkers examined the interactions
However careful selection and chemical modication of of representative gold(III) porphyrins with duplex DNA by
the ligands might allow the respective complexes proper- UVvis absorption titrations [18]. Isosbestic changes and
ties to be eciently tuned with regard to inertness, rate signicant hypochromicity of the Soret band were noticed
of binding to biomolecules and cytotoxic activity. in the electronic spectra of gold(III) porphyrins after addi-
tion of calf-thymus DNA supporting a direct and strong
2. Current views concerning the biochemical actions of interaction with the DNA double helix. Accordingly, in a
cytotoxic gold(III) compounds: a variety of molecular subsequent study the same authors reported that treatment
mechanisms with gold(III) porphyrins induces signicant and character-
istic changes in protein expression proles [23].
As outlined above, the renaissance of interest for In general, these cellular and biochemical studies have
gold(III) compounds as potential anticancer metallodrugs provided further valuable insight into the reactivity and
has resulted, in the course of the last decade, in the synthe- the mode of action of novel gold(III)-based metallodrugs.
sis of a number of structurally diverse gold(III) species, Although the ultimate molecular mechanisms of these
endowed with sucient chemical stability and with relevant new anticancer agents remain largely unknown and elusive,
antiproliferative activities. Detection of in vitro cytotoxicity some considerations and suggestions may be anticipated
has represented, for these metal-based agents, the primary based on the available information.
screening criterion in order to assess their potential anti- Most of the mechanistic studies carried out on cytotoxic
cancer properties. IC50 values of 105 M or lower were gold(III) compounds have been generally referred and
used to indicate a promising, or at least acceptable, anti- compared to the behaviour of cisplatin, for which DNA
tumour ecacy. is one of the major putative targets [24]. However, it
Notably, for a few of these compounds, the pharmaco- emerges from the experimental results collected so far that
logical studies could be extended well beyond assessment the respective molecular mechanisms are rather distinct.
of mere in vitro cytotoxicity by analysing some of the eects Also for cisplatin the major resistance mechanisms fall
they produce at the cellular level such as direct DNA dam- into the following four categories: limitation of drug levels
age, modication of the cell cycle, alterations of mitochon- by reduced uptake and/or increased eux; increased cellu-
drial functions, induction of apoptosis, and so on. lar thiol levels; enhanced DNA repair and/or increased
Moreover, the reactions of some cytotoxic gold(III) damage tolerance; and failure of cell death pathways [25].
complexes with some specic biomolecular targets, e.g. If we consider the cytotoxic eects reported for some of
calf-thymus DNA and a few representative proteins, were the above mentioned gold(III) complexes (Table 1) it is
A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 569
N N N
Cl
Au AuCl4 AuCl3 AuCl2
Cl
N
12 13 14
Au Au Au
Cl2 Ph
(OAc)2
Cl
15 16
17
Me PPh3
NMe2
Au
Me Me
Au
Me
Me
18 19
Fig. 6. Schematic drawings of gold(III) complexes screened for the inhibition of human TrxR1.
family are homodimeric proteins in which each monomer Rigobello in Padova, we have observed that a few gold(III)
includes an FAD prosthetic group, an NADPH binding complexes developed in our laboratory behave as eective
site and an active site containing a redox-active selenol inhibitors of the cytosolic form of the selenoenzyme thiore-
group. Electrons are transferred from NADPH via FAD doxin reductase (TrxR) (see Table 2), accordingly, these
to the active-site selenol of TrxR, which then reduces the compounds were also found to perturb greatly the mito-
substrate [31]. TrxRs are named for their ability to reduce chondrial functions [38].
oxidised thioredoxins (Trxs), a group of small This view is now reinforced by a recent paper by Powis
(10 12 kDa) ubiquitous redox-active peptides which have et al. reporting the inhibitory properties of a series of
a conserved -Trp-Cys-Gly-Pro-Cys-Lys- catalytic site that Au(III) complexes (Fig. 6), against human thioredoxin
undergoes reversible oxidation/reduction of the two Cys reductase 1 (TrxR1), including some gold(III) DAMP com-
residues (see Fig. 5). pounds [39]. Table 2 contains a compilation of representa-
The crystal structure of thioredoxin reductase has been tive literature data for the inhibition of TrxR by various
recently solved [32]. Notably, these structural studies evi- gold compounds.
denced the presence of a selenocysteine group at its active Thus, according to the data described above, it is
site that, in the reduced form, displays a high reactivity reasonable to propose that direct antimitochondrial
toward soft metal ions. There is today sucient evidence eects are the determinant for the large proapoptotic
that this selenol group is the primary anchoring site for a and cytotoxic eects produced by anticancer gold(III)
vast array of metals and metal complexes that are known compounds.
to inhibit thioredoxin reductase [33,34]. Nonetheless, other recent studies identied the thiol-
Thus, one can propose quite reasonably that gold(III) dependent cathepsin enzymes as possible, alternative tar-
compounds exert their cytotoxic eects by causing direct gets for gold-based anticancer agents [40]. These lysosomal
mitochondrial damage through selective modication of enzymes are mainly cysteine proteases responsible for
the selenol active site in thioredoxin reductase as previously extracellular matrix degradation, bone resorption and joint
suggested for auranon and some related gold(I) com- destruction. Their marked inhibition arising from coordi-
pounds [3537]. As a matter of fact, in a recent study con- nation of gold to the active-site cysteine has been reported
ducted in collaboration with the group of Bindoli and for cathespin L and B [41,42].
A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 571
1.2
1.1
1.0
0.9
0.8
0.7
A 0.6 b
0.5
0.4
0.3
0.2
a
0.1
0.0
200 250 300 350 400 450 500 550 600 650 700
nm
0.50
0.45
0.40
0.35 b
0.30
A 0.25
0.20
0.15
0.10
0.05
a
0
200 250 300 350 400 450 500 550 600 650 70 0
nm
Fig. 7. Time dependent spectral proles of Auoxo1cyt c (A) and Auoxo1ubiquitin (B) adducts. Spectra correspond to proteins 105 M before (a) and
after the addition (b) of Auoxo1 in a 1:1 ratio. The further evolution of the various systems over time is reported up to 24 h incubation at 37 C. The buer
was 10 mM phosphate, 20 mM NaCl, pH 7.4.
Very recently a paper on the ecient inhibition of feature for gold compounds. The focus of current research is
cathepsin K and S by two gold(I) derivatives, auranon now on the search of the most important protein targets for
and gold thiomalate, has been published [43]. In this paper gold(III) compounds.
the authors present an X-ray analysis of the gold thioma-
late/cathepsin K complex, showing the selective binding 3. Modelling the interactions of gold(III) compounds with
of gold to the active-site cysteine residue of the protease. protein targets: ESI MS and spectroscopic studies of the
On the ground of the above arguments and of the large reaction of two representative gold(III) complexes with
structural variety of the tested compounds, it can be pro- cytochrome c and ubiquitin
posed that gold(III) complexes produce their biological
eect through a number of distinct biochemical mechanisms In recent years, ESI MS has emerged as an extremely
ranging from classical DNA damage to oxidative membrane powerful tool to monitor, at the molecular level, the forma-
damage, from proteasome inhibition to mitochondrial dam- tion of protein adducts of classical anticancer platinum(II)
age. However, the latter mechanism, i.e. mitochondrial drugs, and to identify the precise nature of the metallic
damage induced through inhibition of thioredoxin reduc- fragments coordinated to protein side chains in these
tase, seems to be a rather peculiar and recurrent mechanistic adducts [4447]. However, only very few ESI MS studies
572 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
953.067
100 885.333
95
90
85
80
826.800
75
70 1032.133
65
Relative Abundance
60
55
50 774.867
45
1125.600
40
35
30
1237.533
25
729.400
1374.467
20
15 1149.733
1545.733
1265.133
10 1274.267
689.200 1409.200
1554.800
5
651.200 1604.333 1772.333
403.133 586.000
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z
953.467
100
885.133
95
1032.400
90
85
80
75
70 1125.867
825.667
65
Relative Abundance
60
55
1238.133
1374.733
50
45
40 1143.867
35
773.800
30 1261.400
25
1390.867 1545.800
1275.733
20 1400.333
15 1416.467
728.400 1423.933
10
1448.333
1564.000
5 716.933 1588.667
681.200 1664.800 1766.333
408.800 519.200
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
m/z
Fig. 8. Multicharged mass spectra of cyt c (A) and Auoxo1cyt c conjugate (B) after 24 h reaction at 37 C.
have dealt so far with protein adducts formed by non-plat- niques, to analyse the reactions of two selected gold(III)
inum anticancer drugs [4850]. compounds, namely compound 7 and its dinuclear ana-
We show here that ESI MS may be advantageously logue Auoxo1, with the model proteins ubiquitin and cyto-
employed, in association with other spectroscopic tech- chrome c. Independent information on these systems was
A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575 573
gained from ICP OES determinations. Overall, the results the free complexes revealed for Auoxo1 peaks at m/z
reported here give us the chance to draw a quite detailed 369.8 and 883.0 which were assigned to [Au(bipy)OH]+
description of the interactions that take place. (100%) and [Au2(bipy)2O2PF6] (14%). For compound 7
First of all, both 7 and Auoxo1 were reacted with either the main peak at m/z 387.4 was assigned to [Au(bi-
ubiquitin or cyt c under identical experimental conditions py)(OH)2]+ and a very weak one at m/z 499.4 to [Au(bi-
and the resulting samples monitored spectrophotometri- py)PF6]+ (3%). It is worth noting that mass spectra of
cally over 24 h at 37 C [21]. proteinmetallodrug samples immediately after mixing
Ubiquitin does not exhibit any absorption band above did not show any adduct formation. This observation indi-
280 nm so that the bands characteristic of the two Au(III) cated that gas phase reactions do not take place under the
compounds may be easily followed. In both cases, the pro- applied experimental conditions.
gressive decrease of the CT bands typical of these gold(III) After 24 h incubation, samples of Auoxo1 and 7 with cyt
complexes is observed, accompanied by the growth of c showed, beyond the signal of the native protein, a charac-
intense bands in the 270300 nm region, characteristic of teristic peak assigned to a 1:1 Aucyt c derivative. The mul-
the free ligand. However, the process is slow, at room tem- ticharged spectra of free cyt c and of its adduct with
perature, and reaches only 30% progression within 24 h. Auoxo1 are reported in Fig. 8. The ESI MS spectra,
On the other hand, horse heart cyt c, in its typical oxi- together with spectrophotometric data, strongly suggest
dised form, manifests intense bands in the visible, namely that upon reaction with the protein the organic ligand is
the Soret band at about 410 nm and broad Q bands lost whereas gold, most likely in the form of a Au+ ion,
between 500 and 550 nm. Nonetheless, the overlap with remains tightly attached to the protein. A similar behav-
the bands characteristic of the two tested gold(III) com- iour was previously reported by Sadler and coworkers in
plexes is not dramatic so that the spectral evolution of the case of the adducts of gold(I) triethylphosphine chlo-
the two distinct components of our system may be moni- ride with cyclophilin [51].
tored rather easily. The reaction of 7 and Auoxo1 with Conversely, ESI MS analysis of the incubation mixtures
cyt c is faster than in the case of ubiquitin. At 24 h the reac- of both complexes with Ub revealed no appreciable adduct
tion has progressed to about 70%. Spectral patterns similar formation even after 12 days of reaction.
to those of ubiquitin are observed showing a continuous In conclusion, the above results point out quite clearly
decrease of the bands of the gold(III) complex and the that independently of the nature of the gold(III) complexes,
growth of an UV band typical of the free ligand. Typical a similar reactivity with cyt c takes place, comprising a
spectral proles obtained for Auoxo1 are shown in Fig. 7. redox process with gold(III) reduction followed by release
Ultraltration experiments accompanied by ICP OES of the organic ligand and eventual coordination of a
determinations showed that a signicant amount of gold gold(I) ion to a protein side chain.
remains associated to cyt c (up to 60% after 24 h). In con- Both the in situ redox process and gold(I) coordination
trast, gold binding to ubiquitin is poor, being only around to a specic protein residue may be crucial events for direct
15% even after several days of incubation. and irreversible protein damage. Further studies are in pro-
Afterward, ESI MS studies were performed on the gress to identify the nature of the binding site for gold(I) in
adducts of 7 or Auoxo1 with either Ub or cyt c. For com- cyt c, good candidates being Met 65 and two accessible his-
parison purposes, all samples were prepared and analysed tidines (His 33 and His 26).
under the identical conditions.1 The proteins, alone, Recent advances in the cellular pharmacology of Au(III)
showed a charge distribution of +7 to +13 and +8 to compounds are leading to the identication of a few spe-
+18 for Ub and cyt c, respectively. ESI MS analyses of cic proteins as probable biomolecular targets. In this
frame it is important to develop appropriate physicochem-
ical methods to monitor reactions of gold(III) complexes
1
Auoxo1 and 7 were dissolved in DMSO and the solution was diluted with protein targets. Modelling work carried out in our
1:100 with H2O to obtain a nal concentration of DMSO of 1%. The laboratory on simple proteins, primarily relying on mass
complexes were incubated at molar ratios of 1:1 with ubiquitin and
cytochrome c in aqueous solution at 37 C yielding nal protein spectrometry and some other ancillary techniques, holds
concentrations of 250 lM. The samples were analysed immediately after much promise for a rapid and detailed characterisation of
start of the incubation, 3 h, 24 h, 48 h, 6 days and 12 days. Before ESI IT goldprotein adducts. Some representative examples have
MS analysis, the incubation solutions were diluted (99:1) with been reported concerning ubiquitin and cytochrome c. Fur-
ACN:H2O:formic acid (30:70:1) and analysed immediately after mixing. ther eorts are needed to extend the investigation to the
Analyses were performed on a Thermo Finnigan LCQ Deca XP Plus
quadrupole ion-trap instrument in positive ion mode. The capillary protein targets that were mentioned in the previous section,
temperature was set at 180 C and the source voltage to 1.50 kV, with a for which, up to now, ESI MS has been scarcely used for
mass range from 300 to 2000. The acquisition was done with Tune Plus 1.3 the characterisation of their interactions with metallodrugs.
SR1 program (Thermo Finnigan) and the deconvolution of data were
performed with Bioworks Browser 3.0 (Thermo Finnigan) on a Windows
4. Concluding remarks
2000 Prof. SR4 PC system using Biomass Calculation and Deconvolution
software. The molecular weight was deconvoluted from the multiply
charged protein and proteinAu adduct peaks with charges ranging from The renewed interest toward gold(III) compounds as
+7 to +13 and +8 to +18 for ubiquitin and cytochrome c, respectively. potential anti-tumour agents, that started at the beginning
574 A. Casini et al. / Journal of Inorganic Biochemistry 102 (2008) 564575
[21] A. Casini, M.A. Cinellu, G. Minghetti, C. Gabbiani, M. Coronnello, [39] L. Engman, M. McNaughton, M. Gajewska, S. Kumar, A. Birming-
E. Mini, L. Messori, J. Med. Chem. 49 (2006) 55245531. ham, G. Powis, Anticanc. Drugs 17 (2006) 539544.
[22] L. Messori, G. Marcon, P. Orioli, M.A. Cinellu, G. Minghetti, Eur. J. [40] V. Turk, B. Turk, D. Turk, EMBO J. 20 (2001) 46294633.
Biochem. 270 (2003) 46554661. [41] A. Chircorian, A.M. Barrios, Bioorg. Med. Chem. Lett. 14 (2004)
[23] Y. Wang, Q.Y. He, C.M. Che, J.F. Chiu, Proteomics 6 (2006) 131 51135116.
142. [42] S.P. Fricker, R. Skerjl, B.R. Cameron, R. Mosi, Y. Zhu, Recent
[24] L.H. Hurley, Nat. Rev. Cancer 2 (2002) 188200. developments in gold drugs in: Contribution to the Gold 2003
[25] D. Wang, S.J. Lippard, Nat. Rev. Drug. Discov. 4 (2005) 307320. Conference: New Industrial Applications for Gold.
[26] M. Coronnello, G. Marcon, S. Carotti, B. Caciagli, E. Mini, T. [43] E. Weidauer, Y. Yasuda, B.K. Biswal, M. Cherny, M.N. James, D.
Mazzei, P. Orioli, L. Messori, Oncol. Res. 12 (2000) 361370. Bromme, J. Biol. Chem. 388 (2007) 331336.
[27] M.T. Coer, C.F. Shaw 3rd, A.L. Hormann, C.K. Mirabelli, S.T. [44] I. Khalaila, C.S. Allardyce, C. Verma, P.J. Dyson, ChemBioChem 6
Crooke, J. Inorg. Biochem. 30 (1987) 177187. (2005) 17881795.
[28] G.F. Rush, P.F. Smith, G.D. Hoke, D.W. Alberts, R.M. Snyder, [45] T. Peleg-Shulman, Y. Najajreh, D. Gibson, J. Inorg. Biochem. 91
C.K. Mirabelli, Toxicol. Appl. Pharmacol. 90 (1987) 391400. (2002) 306311.
[29] C.H. Williams Jr., Eur. J. Biochem. 267 (2000) 6101. [46] A. Casini, G. Mastrobuoni, C. Temperini, C. Gabbiani, S. Francese,
[30] E.S.J. Arner, A. Holmgren, Eur. J. Biochem. 267 (2000) 61026109. G. Moneti, C.T. Supuran, A. Scozzafava, L. Messori, Chem.
[31] H. Nakamura, Antioxid. Redox. Signal. 7 (2005) 823828. Commun. (2007) 156158.
[32] K. Fritz-Wolf, S. Urig, K. Becker, J. Mol. Biol. 370 (2007) 116127. [47] C.G. Hartinger, W.H. Ang, A. Casini, L. Messori, B.K. Keppler, P.J.
[33] A.B. Witte, K. Anestal, E. Jerremalm, H. Ehrsson, E.S. Arner, Free Dyson, JAAS 22 (2007) 960967.
Radic. Biol. Med. 39 (2005) 696703. [48] A.R. Timerbaev, C.G. Hartinger, S.S. Aleksenko, B.K. Keppler,
[34] P.J. Barnard, S.J. Berners-Price, Coord. Chem. Rev. 251 (2007) 1889 Chem. Rev. 106 (2006) 22242248.
1902. [49] A. Casini, G. Mastrobuoni, W.H. Ang, C. Gabbiani, G. Pieraccini,
[35] M.P. Rigobello, G. Scutari, A. Folda, A. Bindoli, Biochem. G. Moneti, P.J. Dyson, L. Messori, Chem. Med. Chem. 2 (2007) 631
Pharmacol. 67 (2004) 689696. 635.
[36] Y. Omata, M. Folan, M. Shaw, R.L. Messer, P.E. Lockwood, D. [50] A. Casini, G. Mastrobuoni, M. Terenghi, C. Gabbiani, E. Monzani,
Hobbs, S. Bouillaguet, H. Sano, J.B. Lewis, J.C. Wataha, Toxicol. In G. Moneti, L. Casella, L. Messori, J. Biol. Inorg. Chem. 12 (2007)
Vitro 20 (2006) 882890. 11071117.
[37] O. Rackham, S.J. Nichols, P.J. Leedman, S.J. Berners-Price, A. [51] J. Zou, P. Taylor, J. Dornan, S.P. Robinson, M.D. Walkinshaw, P.J.
Filipovska, Biochem. Pharmacol. 74 (2007) 9921002. Sadler, Angew. Chem. Int. Ed. Engl. 39 (2000) 29312934.
[38] M.P. Rigobello, L. Messori, G. Marcon, M. Bragadin, A. Folda, G.
Scutari, A. Bindoli, J. Inorg. Biochem. 98 (2004) 16341641.