Reactions of goldIII complexes with serum albuminGiordana Marcon1, Luigi Messori1, Pierluigi Orioli1, Maria Agostina Cinellu2and Giovanni Minghetti2 1 Department of Chemistry, University
Trang 1Reactions of gold(III) complexes with serum albumin
Giordana Marcon1, Luigi Messori1, Pierluigi Orioli1, Maria Agostina Cinellu2and Giovanni Minghetti2
1 Department of Chemistry, University of Florence, Italy; 2 Department of Chemistry, University of Sassari, Italy
The reactions of a few representative gold(III) complexes –
[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2,
[Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2¢,
2¢-terpyridine)Cl]Cl2, [Au(2,2¢-bipyridine)(OH)2][PF6] and
the organometallic compound
[Au(6-(1,1-dimethylbenzyl)-2,2¢-bipyridine-H)(OH)][PF6] – with BSA were investigated
by the joint use of various spectroscopic methods and
separation techniques Weak metal–protein interactions
were revealed for the [Au(ethylenediamine)2]3+ and
[Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas
progressive reduction of the gold(III) centre was observed
in the cases of [Au(2,2¢-bipyridine)(OH)2]+and
[Au(2,2¢,2¢-terpyridine)Cl]2+ In contrast, tight metal–protein adducts
are formed when BSA is reacted with either
[Au(diethylen-triamine)Cl]2+and
[Au(6-(1,1-dimethylbenzyl)-2,2¢-bipyri-dine-H)(OH)]+ Notably, binding of the latter complex to serum albumin results in the appearance of characteristic
CD bands in the visible spectrum It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines Sta-bility of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide The implications of the present results for the pharmacolo-gical activity of these novel cytotoxic agents are discussed Keywords: gold(III) complexes; serum albumin; spectro-scopic measurements
Following the success of platinum(II) compounds in cancer
chemotherapy, several families of nonplatinum metal
complexes have been studied intensely as potential cytotoxic
and antitumour agents In particular, in recent years,
various gold(III) complexes of sufficient stability in the
physiological environment have been prepared and
evalu-ated for in vitro anticancer properties Some of them turned
out to exhibit relevant cytotoxic effects in vitro and were the
subject of further biochemical and pharmacological
inves-tigations [1] Studies of the interactions of these gold(III)
complexes with DNA, the classical target of platinum(II)
complexes, pointed out that binding of these compounds to
nucleic acids is not as tight as in the case of platinum drugs,
suggesting the occurrence of a different mechanism for the
observed biological effects [2,3]
Surprisingly, at variance with the reactions with nucleic
acids, the reactions of antitumour metal complexes with
proteins have been poorly explored until now although they
may be of extreme relevance for the biodistribution, the mechanism of action and the toxic effects of several metallodrugs For example, only a few studies exist on the reactions of the well known anticancer platinum complexes with proteins [4,5]
However, despite the results obtained so far often being incomplete and fragmentary, we believe that the direct damage inflicted on specific proteins by metal complexes, following the formation of strong coordinate bonds, may
be of crucial relevance to explain the biological effects of several metallodrugs, either established clinically or experi-mentally
In the present study, we have considered the reactions of
a series of representative gold(III) complexes, of different structure and of known biological profile, developed in our laboratory, with bovine serum albumin, selected both as the most abundant plasma protein and as a general model for globular proteins Serum albumins have many physiological functions They contribute to colloid osmotic blood pres-sure and are chiefly responsible for the maintenance of blood pH [6] There is evidence of a significant antioxidant activity of serum albumins These molecules may represent the major plasma components that protect against oxidative stress [7] The most outstanding property of albumins is their ability to reversibly bind a large variety of endogenous and exogenous ligands It is worthwhile remembering that serum albumins have often been considered as general ligands for fatty acids, which are otherwise insoluble in blood, and exhibit a high affinity for hematin, bilirubin, and small, negatively charged, hydrophobic molecules; more-over albumins bind various metal ions [8]
The reactions of gold(III) complexes with serum albumin were investigated primarily through the analysis of the
Correspondence to L Messori, Department of Chemistry,
University of Florence, via della Lastruccia, 3, 50019 Sesto
Fiorentino (Florence), Italy.
Fax: + 39 055 4573385, Tel.: + 39 055 4573284,
E-mail: luigi.messori@unifi.it
Abbreviations: en, ethylenediamine (1,2-diaminoethane); dien,
diethy-lentriamine; cyclam, 1,4,8,11-tetraazacyclotetradecane; terpy, 2,2¢,
2¢-terpyridine; bipy, 2,2¢-bipyridine; bipy c
, 6-(1,1-dimethylbenzyl)-2,2¢-bipyridine; CDDP, cis-diammine dichloro platinum(II);
ESI-MS, electrospray ionization mass spectrometry; LMCT,
ligand to metal charge transfer.
(Received 11 July 2003, revised 29 September 2003,
accepted 2 October 2003)
Trang 2characteristic bands of the gold(III) centre in the visible
spectrum Our experiments show that markedly divergent
reactivity patterns with serum albumin have clearly emerged
for the various gold(III) complexes in relation to their
chemical structure and reactivity The implications of such
differences in reactivity are discussed in relation to the
pharmacological properties of the individual compounds
Materials and methods
Materials
[Au(ethylenediamine)2]Cl3 ([Au(en)2]Cl3) was prepared
according to [9] A gummy yellow precipitate was formed
by the addition of a solution of 1,2-ethylendiamine
mono-hydrate in ether to a solution of HAuCl4in ether; the yellow
precipitate was dissolved in water giving an orange solution
A white precipitate of [Au(en)2]Cl3 formed upon adding
ethyl alcohol to the latter solution
[AuCl(diethylentriamine)]Cl2 ([AuCl(dien)]Cl2) was
pre-pared according to [10] A solution of diethylenetriamine/
3HCl in water was added slowly and with stirring to a
solution of HAuCl4 (20%, w/v) and a yellow precipitate
immediately formed A solution of NaOH was added to the
mixture until pH 3 and stirred for 2 h at 0C The yellow
precipitate was then filtered and washed with ethanol
[Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl ([Au
(cyclam)](ClO4)2Cl) was prepared by following the
pro-cedure reported by Kimura et al [11] Treatment of
NaAuCl42H2O with equimolar amounts of cyclam in
CH3CN for 1 h yielded the [Au(cyclam)](ClO4)2Cl complex
[Au(2,2¢,2¢-terpyridine)Cl]Cl2 ([Au(terpy)Cl]Cl2) was
pre-pared by addition of terpyridine to a HAuCl4solution under
a 1 : 1 stoichiometry according to [12]
[Au(2,2¢-bipyri-dine](OH)2][PF6] ([Au(bipy])(OH)2][PF6]) was prepared
according to [13] An aqueous suspension of Ag2O was
added to a solution of [Au(bipy)Cl2][PF6] in acetone The
mixture was stirred for 24 h at room temperature AgCl was
removed by filtration and the solution evaporated to dryness
under reduced pressure The residue was extracted with
acetonitrile and filtered over Celite (Sigma-Aldrich) The
pale-yellow filtrate was concentrated to a small volume and
diethyl ether was added to give a white precipitate of
[Au(bipy)(OH)2][PF6]
An aqueous solution of KOH (33 mg, 0.59 mmol) was
added to an aqueous suspension of [Au(bipyc-H)Cl][PF6]
(179 mg, 0.27 mmol) [14,15] The mixture was refluxed for
1 h under stirring and filtered The volume of the colourless
filtrate was reduced on a rotary evaporator until
crystal-lization was observed The white product [Au(bipyc
-H)(OH)][PF6] was collected by filtration and dried under
vacuum
All the products obtained were checked by elemental
analysis; in all cases, the purity of the compounds was
higher than 98% Further evidence for the correct
identi-fication of the obtained compounds is provided by
electronic spectra and mass spectra (vide infra)
BSA was purchased from Fluka BioChemika (product
number 05470) The powder, lyophilized and crystallized,
was‡ 98.0% pure (purified by HPCE) and of a molecular
mass 66 kDa All the other reagents, purchased
from Sigma-Aldrich, were of analytical grade Where
not stated otherwise, experiments were performed in phosphate buffer containing 50 mM Na2HPO4, 100 mM NaCl, pH 7.4
Spectroscopic measurements The interaction of all complexes with BSA was analysed by monitoring the electronic spectra of freshly prepared solutions of each complex after mixing with BSA (in the ratio 1 : 1) in the reference buffer The concentration of [Au(en)2]Cl3, [Au(dien)Cl]Cl2 and [Au(cyclam)](ClO4)2Cl was 1· 10)3M, while [Au(terpy)Cl]Cl2 was 1· 10)4M, [Au(bipy)(OH)2][PF6] and [Au(bipyc-H)(OH)][PF6] 2.25· 10)4M Visible absorption spectra were carried out with a PerkinElmer Lambda Bio 20 spectrophotometer The measurements were done at room temperature (25C) Fluorescence spectra were registered with a Jasco
FP-750 spectrofluorimeter working at room temperature with
kex¼ 295 nm; BSA 5 · 10)5M was titrated with
[Au(bi-pyc-H)(OH)][PF6] at the ratios [Au(bipyc-H)(OH)][PF6]/ BSA r¼ 0.5–5.0 (where r is moles of drug per mole of BSA)
Ultrafiltration experiments The adducts between gold(III) compounds and BSA, prepared as described above, were filtered after 24 h incubation at room temperature, using Centricon YM-10 (Amicon Bioseparations, Millipore Corporation, USA) at
1370 g and the starting volume reduced by half; finally, the absorption spectra of the upper and lower portions of the solution were recorded
Extensive ultrafiltration was applied to the same samples and the absorption spectra were recorded after three cycles
of washing with the buffered solution
Additional experiments were conducted by ultracentri-fuging at half volume [Au(dien)Cl]Cl2/BSA solutions at molar ratios of 1 : 1, 2 : 1, 4 : 1 and 8 : 1 Complex content
in the upper and lower solution was analysed spectro-photometrically
Circular dichroism spectra
CD spectra of BSA samples at increasing [Au(bipyc -H)(OH)][PF6]/BSA molar ratios, in phosphate buffer, were recorded on a Jasco J500Cdichrograph and analysed through the standardJASCOsoftware The time dependence
of the spectra was analysed over several hours; the final spectra were recorded after 24 h incubation at 25C Reaction with cyanide
[Au(dien)Cl]Cl2/BSA and [Au(bipyc-H)(OH)][PF6]/BSA adducts were treated with a 10 : 1 stoichiometric excess of cyanide The UV-Vis spectra were recorded before and immediately after the addition of a concentrated solution
of sodium cyanide
Reaction with imidazole The interaction of [Au(bipyc-H)(OH)][PF6] 2.5· 10)4M and [Au(dien)Cl]Cl 1· 10)3Mwith imidazole (in the ratio
Trang 31 : 1) was analysed by monitoring the electronic spectra of a
freshly prepared solutions in the reference buffer at 25C,
5 h long
Results
Structure and solution chemistry of the investigated
gold(III) complexes
In the present study we have considered the following six
gold(III) complexes: [Au(en)2]Cl3, [Au(dien)Cl]Cl2,
[Au(cy-clam)](ClO4)2Cl, [Au(terpy)Cl]Cl2, [Au(bipy)(OH)2][PF6]
and [Au(bipyc-H)(OH)][PF6], recently investigated in our
laboratory (Fig 1) The choice of these gold(III) complexes
was dictated by their favourable chemical properties in
terms of solubility in water and stability within a
physio-logical-like environment; in addition, most of these
com-plexes are endowed with relevant cytotoxic properties
toward cultured human tumour cell lines, as previously
reported The solution behaviour of these complexes, within
a reference physiological buffer, was further assayed by
monitoring the characteristic visible bands over several
hours An appreciable stability was revealed for all
men-tioned gold(III) complexes in line with previous reports [1,3]
Spectrophotometric studies of the reaction with BSA
As all these gold(III) complexes, under physiological
conditions, exhibit intense and characteristic charge transfer
bands in the visible, their reactions with BSA were monitored directly by visible absorption spectroscopy BSA was added in 1 : 1 stoichiometric amounts to buffered solutions of each gold(III) complex and the visible spectra of the resulting mixture recorded over several hours at room temperature The obtained spectrophotometric patterns are shown in Fig 2
Different behaviours clearly emerge from direct inspec-tion of the spectral profiles It is apparent that the spectra of either [Au(en)2]Cl3or [Au(cyclam)](ClO4)2Cl are not signi-ficantly affected by addition of BSA These observations suggest that the gold(III) chromophore of these complexes
is not – or is only slightly – perturbed by protein addition Small changes are observed in the main charge transfer band for both [Au(dien)Cl]Cl2 and [Au(bipyc-H)(OH) ][PF6] For [Au(dien)Cl]Cl2, the changes are complete within about 2 h, while only a few minutes are needed in the case of [Au(bipyc-H)(OH)][PF6]
In contrast, in the cases of [Au(terpy)Cl]Cl2 and [Au(bipy)(OH)2][PF6], a progressive decrease in intensity
of the visible bands is observed until complete disappear-ance Under the experimental conditions that we have used, the process is complete within 2 h in the case of [Au(terpy)Cl]Cl2 and within about 6 h in the case of [Au(bipy)(OH)2][PF6] After ultrafiltration of the adducts between BSA and [Au(terpy)Cl]Cl2 or [Au(bipy)(OH)2] [PF6], the lower solutions were spectrophotometrically analysed and found to contain the free ligands terpyridine and bipyridine No gold was detected in these solutions As these gold(III) complexes are fairly stable, the best explan-ation of the above observexplan-ation is that gold(III) undergoes reduction and the complexes break down with release of the ligands In turn, gold may be reduced to gold(I) or even to colloidal gold associated with the protein
Adduct formation as assessed by ultrafiltration experiments
Further information on the reactions of gold(III) complexes with BSA was gained by the application of classical biochemical separation techniques The main goal of these experiments was to provide at least qualitative information
on the strength of the interactions between gold(III) complexes and BSA Buffered solutions of the individual gold(III) complexes and BSA were prepared, at 1 : 1 stoichiometry, and incubated for 12–24 h at room tempera-ture Ultrafiltration with a Centricon device was carried out
to reduce sample volumes from 2 to 1 mL, and the upper and lower solutions analysed spectrophotometrically We noticed that [Au(en)2]Cl3 and [Au(cyclam)](ClO4)2C l are readily removed from the protein by ultrafiltration, imply-ing that the interaction is relatively weak and most likely electrostatic in nature Figure 3A shows the results obtained with [Au(cyclam)](ClO4)2Cl) In the case of [Au(bipy)(OH)2][PF6] (Fig 3B), disruption of the gold(III) complex is confirmed by the appearance of the characteristic UV-Vis bands of the free ligand 2,2¢-bipyridine (at 230 and
280 nm) in the lower solution after ultrafiltration
In contrast, both [Au(dien)Cl]Cl2 and [Au(bipyc -H)(OH)][PF6] are not easily displaced from the protein For [Au(dien)Cl]Cl2, the protein-bound complex after a single ultrafiltration is about 80%, while for [Au(bipyc
-Fig 1 Schematic drawings of some representative gold(III) complexes.
[Au(en) 2 ]Cl 3 , [Au(dien)Cl]Cl 2 , [Au(cyclam)](ClO 4 ) 2 Cl, [Au(terpy)Cl]
Cl , [Au(bipy)(OH) ][PF ] and [Au(bipyc-H)(OH)][PF ].
Trang 4H)(OH)][PF6] it is more than 96%, suggesting that these
complexes are tightly bound to BSA through coordinate
bonds However, [Au(dien)Cl]Cl2 may be removed by
repeated cycles of ultrafiltration while [Au(bipyc
-H)(OH)][PF6] is not Representative results of repeated
ultrafiltration experiments are shown in Fig 4
The [Au(dien)Cl]Cl2/BSA system
The appreciable stability of the [Au(dien)Cl]Cl2/BSA
adducts prompted us to analyse this system in more detail
Specifically, we tested whether protein binding is reversible
and whether multiple binding sites are available for
[Au(dien)Cl]Cl2 on the protein surface To address these
issues, [Au(dien)Cl]Cl2/BSA solutions were prepared at
molar ratios of 1 : 1, 2 : 1, 4 : 1 and 8 : 1; the gold content
in the upper and lower solutions was analysed
spectropho-tometrically after extensive ultracentrifugation From
ana-lysis of the experimental results, it is apparent that the
relative percentage of bound gold decreases as the
[Au(dien)Cl]Cl2/BSA ratio increases (Table 1) When BSA
is exposed to an 8 : 1 [Au(dien)Cl]Cl2molar excess, about
2.4 gold atoms are found associated with each protein
molecule after extensive washing Overall, these findings
suggest that multiple binding sites for [Au(dien)Cl]Cl2 are
present on BSA, of progressively lower affinity
CD spectrum of the [Au(bipyc-H)(OH)][PF6]/BSA adduct
Further information on the spectral features of BSA-bound
gold(III) centres was obtained by CD spectroscopy, a
particularly well-suited technique to analyse the specific environment of protein-bound metal centres [16]
A sample of [Au(bipyc-H)(OH)][PF6]/BSA was prepared
at a 1 : 1 molar ratio, and analysed by CD, immediately after mixing, at 25C(Fig 5) Notably this adduct is characterized by an intense CD negative band in the visible spectrum, at k¼ 410 nm, diagnostic of the fact that the gold(III) species is bound to a chiral matrix such as the protein
With [Au(dien)Cl]Cl2, only minor modifications were observed in the CD spectra of 10)4M BSA when the gold(III) complex was added in the ratios 1 : 1, 2 : 1, 4 : 1 and 8 : 1; however, no clear characteristic CD band appeared in the visible spectrum (data not shown) Gold removal from BSA by potassium cyanide
To further assess the stability of the adducts, either [Au(dien)Cl]Cl2/BSA and [Au(bipyc-H)(OH)][PF6]/BSA were treated with a 10 : 1 stoichiometric excess of cyanide
It is well known that excess cyanide leads to the formation
of a very stable tetracyanoaurate complex and we therefore wanted to check whether such a strong ligand is able to remove gold(III) from the protein, both kinetically and thermodynamically Indeed, treatment with cyanide results
in quick disappearance of the peculiar visible bands of the gold(III) centres in both adducts implying that the bound gold is accessible and that the kinetics of release are fast In contrast, treatment of these derivatives with lower amounts
of cyanide did not result in complete detachment of gold from the protein
Fig 2 Time-dependent spectral profiles of gold(III) compounds/BSA adducts Visible absorption spectra of buffered solutions con-taining gold(III) complexes and BSA in a 1 : 1 ratio Spectra correspond to [Au(en) 2 ]Cl 3
1 · 10)3M (A), [Au(dien)Cl]Cl 2 1 · 10)3M (B), [Au(cyclam)](ClO 4 ) 2 C l 1 · 10)3M (C), [Au(terpy)Cl]Cl 2 1 · 10)4M (D), [Au(bipy) (OH) 2 ][PF 6 ] 2.25 · 10)4M (E) and [Au(bipy c -H)(OH)][PF 6 ] 2.25 · 10)4M (F), before (a) and after the addition of BSA The further evolution of the various systems over time is reported until the spectral changes reach completion The buffer (pH 7.4) con-tains 50 m M Na 2 HPO 4 and 100 m M NaCl.
Trang 5Surface histidines as the probable binding site
for gold(III) complexes: the reaction with imidazole
Imidazoles of surface histidines are good candidates as
donors for the gold(III) centre To elucidate this issue we
carried out the reaction of [Au(dien)Cl]Cl2and [Au(bipyc
-H)(OH)][PF6] with imidazole, within the same buffer, and
analysed the modifications of the visible spectra of the
gold(III) chromophore Interestingly, spectral changes
similar to those observed upon reaction of the same
complex with albumin were detected This observation,
although not conclusive, favours the view that histidines
are the probable binding sites for the gold(III) containing
fragments
Fluorescence studies
Fluorescence measurements give information about the
molecular environment in the vicinity of the chromophore
molecules The intensity of intrinsic fluorescence of two
tryptophan residues (Trp213 and Trp314) and a shift in
wavelength of their emission maxima were chosen as
indicators of protein conformational changes in serum
albumin
Notably, the addition of [Au(bipyc-H)(OH)][PF6] to
BSA-buffered solutions results in a net decrease of
fluores-cence intensity; indeed, progressive fluoresfluores-cence quenching
is observed as the [Au(bipyc-H)(OH)][PF]/BSA molar ratio
increases from 0.5 to 5 (Fig 6) At higher ratios, saturation
is reached and the final fluorescence spectrum is assigned
to the protein-bound form of [Au(bipyc-H)(OH)][PF6] Whereas the residual fluorescence intensity is only 5%
of the original value, the position of the maximum moved toward red wavelengths (a Dk¼+13 nm has been deter-mined for r¼ 5)
The shift in the position of the emission maximum corresponds to the changes of the polarity around the chromophore molecule The slight red-shift observed indi-cates that tryptophan residues were placed in a more polar environment and were more exposed to the solvent It is possible that [Au(bipyc-H)(OH)][PF6] sticks to BSA mole-cules and consequently rearranges the tryptophan micro-environment
Fig 4 The exhaustive ultrafiltration experiments of two representative gold(III) compounds/BSA adducts Visible absorption spectra of the adduct before (a) and after exhaustive ultrafiltration: the spectra of the lower (l) and the upper (u) solutions are shown These data refer to the [Au(dien)Cl]Cl 2 /BSA (A) and [Au(bipy c -H)(OH)][PF 6 ]/BSA (B) adducts (1 : 1).
Fig 3 The ultrafiltration experiments at half volume of two
represen-tative gold(III) compounds/BSA adducts Visible absorption spectra of
the lower (l) and upper (u) solution obtained after ultrafiltration
(reducing the volume to half) These data refer to the
[Au(cy-clam)](ClO 4 ) 2 Cl/BSA (A) and [Au(bipy)(OH) 2 ][PF 6 ]/BSA (B) adducts
(1 : 1).
Table 1 Percentages of [Au(dien)Cl]Cl 2 in the upper and lower solutions after ultracentrifugation Percentage of complex found in the upper and lower fractions after ultracentrifugation of solutions containing the [Au(dien)Cl]Cl 2 /BSA system in the ratios 1 : 1, 2 : 1, 4 : 1 and 8 : 1 Fraction 1 : 1 2 : 1 4 : 1 8 : 1 Upper solution 70.3 60.0 46.3 31.8 Lower solution 29.7 40.0 53.7 68.2
Trang 6Biological properties of the adduct
[Au(bipyc-H)(OH)][PF6]/BSA 1 : 1
It is still a matter of debate whether protein adducts of
cytotoxic metallodrugs retain, at least in part, the
anti-tumour properties of the free metal complex In order to
address this point the biological activity of the adduct
[Au(bipyc-H)(OH)][PF6]/BSA 1 : 1 was tested toward some
representative human tumour cell lines We observed that
the adduct retained to a good extent the cytotoxic activity of
the free metal complex; probably the protein behaves as a
ÔreservoirÕ of the free gold(III) compound (Table 2)
Discussion
The reactions of anticancer metal complexes with proteins
have been scarcely investigated until now We believe that
this issue is of particular relevance in view of the established
reactivity of metal complexes with model proteins, and
deserves, in any case, greater attention In fact, metal–protein interactions may play key roles in the biodistribution, in the mechanism of action and in the toxic effects of antitumour metal complexes Moreover, this subject is becoming more important because the paradigm that DNA is a primary target for antitumour metallodrugs is rapidly declining, and seems to be no longer valid, at least for some families of nonplatinum anticancer metal complexes Obviously, this observation has prompted new interest in the search of novel proteins as possible targets for such metallodrugs
Even in the case of cisplatin, the knowledge of the interactions with proteins is limited to a few studies only, from which, notwithstanding, it emerges that the largest portion of administered platinum is associated with pro-teins Cole reported that cisplatin binds in vitro almost irreversibly to BSA [17]; due to the apparent irreversibility (both in vivo and in vitro) of the protein/195mPt–cisplatin complex, it is unlikely that the protein-bound fraction of the administered free drug will serve as a therapeutically useful drug reservoir [18]
Other studies have been reported on the interactions of some well known anticancer ruthenium(III) complexes and
of auranofin with plasma proteins [19–21]
Very scarce information exists on the reaction of gold(III) complexes with proteins In fact gold(III) complexes gen-erally behave as strong oxidizing agents; hence it is commonly believed that they are quickly reduced to gold(I) compounds or to colloidal gold by low molecular mass biomolecules and by protein side chains
Thus, in the present paper, we have tried to detail the reactions of a series of emerging antitumour gold(III) complexes of appreciable redox stability with serum albu-min, used as a general model for globular proteins In the compounds investigated the oxidizing properties of the gold(III) centre are drastically decreased by the presence of strong multidentate ligands in such a way that interaction studies are feasible However, the stronger oxidizing agents
in our series ([Au(terpy)Cl]Cl2 and [Au(bipy)(OH)2][PF6]) are still able to slowly oxidize the protein side chains At variance with this, the complexes with less pronounced oxidizing properties do not give rise to significant redox chemistry but tend to form adducts with BSA that appear to
be of different strength The tight adducts that formed with either [Au(dien)Cl]Cl2 or [Au(bipyc-H)(OH)][PF6] were further investigated Compared to the cisplatin–BSA adduct, the adduct between the organometallic gold(III)
Fig 5 Circular dichroism spectra of the [Au(bipyc-H)(OH)][PF 6 ]/BSA
adduct Circular dichroism spectra of BSA and of the [Au(bipyc
-H)(OH)][PF 6 ]/BSA adduct in the 1 : 1 ratio The spectrum of the
adduct was recorded immediately after mixing and after 3 h BSA
concentration was 2 · 10)4M
Fig 6 Titration of BSA with [Au(bipy c -H)(OH)][PF 6 ] studied by
fluorescence Fluorescence spectra of 5 · 10)5M BSA upon addition of
increasing amounts of [Au(bipy c -H)(OH)][PF 6 ], in the reference buffer
are shown In the course of the experiment, r varies from 0.5 to 5.0.
Table 2 Cytotoxic activity of [Au(bipyc-H)(OH)][PF 6 ] and of its adduct with BSA Inhibitory effects of [Au(bipyc-H)(OH)][PF 6 ], the adduct Au(bipy C -H)(OH)][PF 6 ]/BSA and cisplatin on the growth of some cisplatin-sensitive (A2780/S) and -resistant (A2780/R, SKOV3) human tumour cell lines ED 50 is defined as the concentration of drug required
to inhibit cell growth by 50% compared to control.
Cell line
ED 50 (l M ) [Au(bipyC -H)(OH)][PF 6 ]
[Au(bipyC -H)(OH)][PF 6 ]/BSA cisplatin
Trang 7compound and BSA, once formed, is stable and retains its
cytotoxic activity; in other words it seems to be a good
candidate for further pharmacological evaluation Notably,
the main features of the gold(III) centre are conserved after
association with BSA The adducts are relatively stable and
may be destroyed only by the addition of strong ligands for
gold(III) such as cyanide
This behaviour is interpreted in terms of either weak
electrostatic interactions or direct metal coordination to
surface residues of the protein The ability of selected
complexes to tag either cysteine or histidine residues may
result in specific damaging of crucial proteins, which could
account for the pharmacological and toxic effects Some
reports exist in the literature indicating that histidine
residues are preferred binding sites for ruthenium(III) on
the protein surface [22,23] The antiarthritic gold(I) drug
Auranofin is known to bind specifically Cys34 of human
serum albumin [24] In the light of the above examples it
might well be that selective modification of surface protein
residues by gold(III) complexes constitutes the molecular
basis for their biological effects
Concluding remarks
In this study we have investigated the reactions of six
representative gold(III) complexes with bovine serum
albu-min used as a general model for plasma proteins Different
patterns of reactivity emerge for the various compounds in
relation to the specific chemical properties of the gold(III)
complexes In some cases tight adducts are formed in which
the bound gold(III) centres are probably coordinated to
surface histidines of the protein It is hypothesized that the
ability of selected gold(III) complexes to tag either cysteine
or histidine residues may result in specific damaging of
crucial intracellular proteins thus accounting for the
relevant cytotoxic effects of these compounds
Acknowledgements
The Cassa di Risparmio di Firenze and MIUR are gratefully
acknowledged for a generous grant We thank Dr Costanza Landi
and Alessandro Vaccini for helping us in the experimental work.
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