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A novel gold(I) complex inspired by the known medicinal inorganic compounds auranofin and thimerosal, namely ethylthiosalicylate(triethylphosphine)gold(I) (AFETT hereafter), was synthesized and characterised and its structure was resolved through X-ray diffraction. The solution behavior of AFETT and its interactions with two biologically relevant proteins (

The use of Pt(iv) complexes as potential anticancer drugs is attractive, because they have higher stability and less side effects than Pt(ii) compounds. Moreover, some Pt(iv) complexes can also be activated with light, opening an avenue to photochemotherapy. Our purpose is to widen the library of photoactivatable Pt(iv)-based prodrugs and here we report on the oxidation of the Pt(ii) compound [PtCl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (1) with PhICl2 or H2O2. The synthetic procedure avoids the formation of multiple species: the treatment with PhICl2 produces the Pt(iv) complex with axial chlorides, [PtCl3(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (2), while H2O2 oxidation and post-synthesis carboxylation produce [Pt(OCOCH3)2Cl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (3), bearing acetates in the axial positions. 2 and 3 are stable in physiological-like buffers and in DMSO in the dark, but undergo photoreduction to 1 upon irradiation at 365 nm. Their stability toward reduction is a fundamental parameter to consider: cyclic voltammetry experiments show that the 2 electron reduction Pt(iv) → Pt(ii) occurs at a more negative potential for 3, because of the greater stabilization provided by the acetate axial groups; noteworthily, 3 is stable for hours also in the presence of mM concentration of glutathione. The cytotoxicity of 2 and 3 toward A2780 and A2780cis cell lines reveals that 3 is the least toxic in the dark, but is able to produce cytotoxic effects far higher than cisplatin when irradiated. To shed light on the mechanistic aspects, the interaction with protein and DNA models has been explored through high-resolution mass spectrometry revealing that 2 and 3 behave as prodrugs, but are able to bind to biological targets only after irradiation.

Quite surprisingly, cisplatin and cis-[PtI

In this work, we have analysed the binding of the Pt(II) complexes ([PtCl(4′-phenyl-2,2′:6′,2″-terpyridine)](CF3SO3) (1), [PtI(4′-phenyl-2,2′:6′,2″-terpyridine)](CF3SO3) (2) and [PtCl(1,3-di(2-pyridyl)benzene) (3)] with selected model proteins (hen egg-white lysozyme, HEWL, and ribonuclease A, RNase A). Platinum coordination compounds are intensively studied to develop improved anticancer agents. In this regard, a critical issue is the possible role of Pt-protein interactions in their mechanisms of action. Multiple techniques such as differential scanning calorimetry (DSC), electrospray ionization mass spectrometry (ESI-MS) and UV-Vis absorbance titrations were used to enlighten the details of the binding to the different biosubstrates. On the one hand, it may be concluded that the affinity of 3 for the proteins is low. On the other hand, 1 and 2 strongly bind them, but with major binding mode differences when switching from HEWL to RNase A. Both 1 and 2 bind to HEWL with a non-specific (DSC) and non-covalent (ESI-MS) binding mode, dominated by a 1:1 binding stoichiometry (UV-Vis). ESI-MS data indicate a protein-driven chloride loss that does not convert into a covalent bond, likely due to the unfavourable complexes’ geometries and steric hindrance. This result, together with the significant changes of the absorbance profiles of the complex upon interaction, suggest an electrostatic binding mode supported by some stacking interaction of the aromatic ligand. Very differently, in the case of RNase A, slow formation of covalent adducts occurs (DSC, ESI-MS). The reactivity is higher for the iodo-compound 2, in agreement with iodine lability higher than chlorine.

Metal complexes of N-heterocyclic carbene (NHC) ligands are the object of increasing attention for therapeutic purposes. Among the different metal centres, interest on Au-based compounds started with the application as anti-arthritis drugs. On the other hand, Ag(I) antimicrobial properties have been known for a long time. For Au(I)/Au(III)-NHC and Ag(I)-NHC anti-tumour and anti-proliferative properties have been quite recently demonstrated. In addition to these and as for Group 11, copper is a much less investigated metal centre, but a few papers underline its pharmacological potential. This review wants to focus on the different biological targets for these metal-based compounds. It is divided into chapters which are respectively devoted on: i) mitochondria and thiol oxidoreductase systems; ii) other relevant enzymes; iii) nucleic acids. Examples of representative coinage NHCs for each of the targets are provided together with significant references on recent advances on the topic. Moreover, a final comment summarises the aspects enlightened by each chapter and provides some hints to better understand the metal-NHCs mechanistic behaviour based on structure-activity relationships.

The trinuclear platinum cluster [Pt3(μ-PBut2)3(CO)3]CF3SO3 (I) was designed featuring the presence of a nearly equilateral platinum triangle bridged by three di-tert-butylphosphide ligands; in addition, each platinum center bears a terminal carbonyl ligand. This triplatinum cluster was initially developed in view of applications in the field of cluster-containing innovative materials. Yet, due to the large success of platinum complexes in cancer treatment, we also decided to explore its cytotoxic and anticancer properties. Accordingly, the solubility profile of this compound in several solvents was preliminarily investigated, revealing a conspicuous solubility in DMSO and DMSO/buffer mixtures; this makes the biological testing of I amenable. UV–Vis measurements showed that the triplatinum cluster is stable for several hours under a variety of conditions, within aqueous environments. No measurable reactivity was observed for I toward two typical model proteins, i.e. lysozyme and cytochrome c. On the contrary, a significant reactivity was evidenced when reacting I with small sulfur-containing ligands. In particular, a pronounced reactivity with reduced glutathione and cysteine emerged from ESI-MS experiments, proving complete formation of I-GSH and I-Cys derivatives, with the loss of a single carbonyl ligand. Starting from these encouraging results, the cytotoxic potential of I was assayed in vitro against a panel of representative cancer cell lines, and potent cytotoxic properties were disclosed. Of particular interest is the finding that the triplatinum species manifests potent antiproliferative properties toward Triple Negative Breast Cancer Cells, often refractory to most anticancer drugs. Owing to the reported encouraging results, a more extensive biological and pharmacological evaluation of this Pt cluster is now warranted to better elucidate its mode of action.

The bis carbene gold(I) complex [Au(1-butyl-3-methyl-2-ylidene)2]PF6, ([Au(NHC)2]PF6 hereafter), holds remarkable interest as a perspective anticancer agent. The compound is stable under physiological like conditions: its original structure is retained even in the presence of excess glutathione (GSH). Previous studies revealed its high cytotoxicity in vitro that correlates with the impairment of crucial metabolic and enzymatic cellular processes (Magherini et al., Oncotarget, 2018, 9, 28042). Here, the interaction of [Au(NHC)2]PF6 with the human telomeric DNA G-quadruplex Tel23 has been investigated in solution by means of high resolution mass spectrometry. ESI MS experiments well document the formation of stable 1 : 1 adducts between the biscarbene gold complex – in its intact form – and the DNA G-quadruplex Tel23. Next, through independent biophysical methods, we show that [Au(NHC)2]PF6 binding does not significantly affect the G quadruplex melting temperature nor its conformation. The crystal structure for the [Au(NHC)2]+/Tel24 adduct was eventually determined by a joint X-ray diffraction and in silico simulation approach. Through the careful integration of solution and solid-state data, a quite clear picture emerges for the interaction of this gold complex with the Tel23 G-quadruplex.

The antiproliferative properties of a group of 13 structurally diverse gold(III) compounds, including six mononuclear gold(III) complexes, five dinuclear oxo-bridged gold(III) complexes, and two organogold(III) compounds, toward several human tumor cell lines were evaluated in vitro using a systematic screening strategy. Initially all compounds were tested against a panel of 12 human tumor cell lines, and the best performers were tested against a larger 36-cell-line panel. Very pronounced antiproliferative properties were highlighted in most cases, with cytotoxic potencies commonly falling in the low micromolar—and even nanomolar—range. Overall, good-to-excellent tumor selectivity was established for at least seven compounds, making them particularly attractive for further pharmacological evaluation. Compare analysis suggested that the observed antiproliferative effects are caused by a variety of molecular mechanisms, in most cases "DNA-independent,” and completely different from those of platinum drugs. Remarkably, some new biomolecular systems such as histone deacetylase, protein kinase C/staurosporine, mammalian target of rapamycin/rapamycin, and cyclin-dependent kinases were proposed for the first time as likely biochemical targets for the gold(III) species investigated. The results conclusively qualify gold(III) compounds as a promising class of cytotoxic agents, of outstanding interest for cancer treatment, while providing initial insight into their modes of action. Graphical Abstract: A series of gold(III) compounds showed cytotoxic properties and tumor selectivity toward a panel of cancer cell lines. Compare analysis provided insight into their possible mechanisms of action

The triplatinum complex of the 2,4,6-Tris(2-pyrimidyl)-1,3,5-triazine ligand, Pt

A series of organometallic AuI N-heterocyclic carbene (NHC) complexes was synthesized and characterized for anticancer activity in four human cancer cell lines. The compounds' toxicity in healthy tissue was determined using precision-cut kidney slices (PCKS) as a tool to determine the potential selectivity of the gold complexes ex vivo. All evaluated compounds presented cytotoxic activity toward the cancer cells in the nano-or low micromolar range. The mixed AuI NHC complex, (tert-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl) imidazol-2-ylidene gold(I), bearing an alkynyl moiety as ancillary ligand, showed high cytotoxicity in cancer cells in vitro, while being barely toxic in healthy rat kidney tissues. The obtained results open new perspectives toward the design of mixed NHC-alkynyl gold complexes for cancer therapy.

Aubipyc is an organogold(III) compound endowed with encouraging anti-proliferative properties in vitro that is being evaluated pre-clinically as a prospective anticancer agent. A classical proteomic approach is exploited here to elucidate the mechanisms of its biological actions in A2780 human ovarian cancer cells. Based on 2-D gel electrophoresis separation and subsequent mass spectrometry identification, a considerable number of differentially expressed proteins were highlighted in A2780 cancer cells treated with Aubipyc. Bioinformatic analysis of the groups of up-regulated and down-regulated proteins pointed out that Aubipyc primarily perturbs mitochondrial processes and the glycolytic pathway. Notably, some major alterations in the glycolytic pathway were validated through Western blot and metabolic investigations. Biological significance This is the first proteomic analysis regarding Aubipyc cytotoxicity in A2780/S ovarian cancer cell line. Aubipyc is a promising gold(III) compound which manifests an appreciable cytotoxicity toward the cell line A2780, being able to overcome resistance to platinum. The proteomic study revealed for Aubipyc different cellular alterations with respect to cisplatin as well as to other gold compound such as auranofin. Remarkably, the bioinformatic analysis of proteomic data pointed out that Aubipyc treatment affected, directly or indirectly, several glycolytic enzymes. These data suggest a new mechanism of action for this gold drug and might have an impact on the use of gold-based drug in cancer treatment.

A group of mixed-ligand Pt(II) complexes bearing acetylacetonate and sulphur ligands were recently developed in the University of Lecce as a new class of prospective anticancer agents that manifested promising pharma-cological properties in preliminary in vitro and in vivo tests. Though modelled on the basis of cisplatin, these Pt(II) complexes turned out to exhibit a profoundly distinct mode of action as they were found to act mainly on non-genomic targets rather than on DNA. Accordingly, we have explored here their reactions with two representative model proteins through an established ESI–MS procedure with the aim to describe their general interaction mechanism with protein targets. A pronounced reactivity with the tested proteins was indeed documented; the nature of the resulting metallodrug-protein interactions could be characterised in depth in the various cases. Preferential binding to protein targets compared to DNA is supported by independent ICP-OES measurements. The implications of these findings are discussed.

In the last few years gold(III) complexes have attracted growing attention in the medicinal chemistry community as candidate anticancer agents. In particular some organogold(III) compounds manifested quite attractive pharmacological behaviors in preclinical studies. Here we compare the chemical and biological properties of the novel organogold(III) complex [Au(bipy(dmb)-H)(NH(CO)CH3)][PF6] (Aubipy(aa)) with those of its parent compounds [Au(bipy(dmb)-H)(OH)][PF6] (Aubipy(c)) and [Au2(bipy(dmb)-H)2)(μ-O)][PF6]2 (Au2bipy(c)), previously synthesized and characterized. The three study compounds were comparatively assessed for their antiproliferative actions against HCT-116 cancer cells, revealing moderate cytotoxic effects. Proapoptotic and cell cycle effects were also monitored. Afterward, to gain additional mechanistic insight, the three gold compounds were challenged against the model proteins HEWL, RNase A and cytochrome c and reactions investigated through UV-Vis and ESI-MS analysis. A peculiar and roughly invariant protein metalation profile emerges in the three cases consisting of protein binding of {Au(bipy(dmb)-H)} moieties. The implications of these results are discussed in the frame of current knowledge on anticancer gold compounds.

The dibromido analogue of cisplatin, cis-PtBr2(NH3)2 (cisPtBr2 hereafter), has been prepared and characterised. Its solution behaviour in standard phosphate buffer, at pH 7.4, was investigated spectrophotometrically and found to reproduce quite closely that of cisplatin; indeed, progressive sequential release of the two halide ligands typically occurs as in the case of cisplatin, with a roughly similar kinetics. Afterward, patterns of reactivity toward model proteins and standard ctDNA were explored and the nature of the resulting interactions elucidated. The antiproliferative properties were then evaluated in four representative cancer cell lines, namely A549 (human lung cancer), HCT116 (human colon cancer), IGROV-1 (human ovarian cancer) and FLG 29.1 (human acute myeloid leukaemia). Cytotoxic properties in line with those of cisplatin were highlighted. From these studies an overall chemical and biological profile emerges for cisPtBr2 closely matching that of cisplatin; the few slight, but meaningful differences that were underscored might be advantageously exploited for clinical application.

Gold(III) compounds form a family of promising cytotoxic and potentially anticancer agents that are currently undergoing intense preclinical investigations. Four recently synthesized and characterized gold(III) derivatives of 2-substituted pyridines are evaluated here for their biological and pharmacological behavior. These include two cationic adducts with 2-pyridinyl-oxazolines, [Au(pyox(R))Cl(2)][PF(6)], [pyox(R)=(S)-4-benzyl-2-(pyridin-2-yl)-4,5-dihydrooxazole, I; (S)-4-iso-propyl-2-(pyridin-2-yl)-4,5-dihydrooxazole, II] and two neutral complexes [Au(N,N'OH)Cl(2)], III, and [Au(N,N',O)Cl], IV, containing the deprotonated ligand N-(1-hydroxy-3-iso-propyl-2-yl)pyridine-2-carboxamide, N,N'H,OH, resulting from ring opening of bound pyox(R) ligand of complex II by hydroxide ions. The solution behavior of these compounds was analyzed. These behave as classical prodrugs: activation of the metal center typically takes place through release of the labile chloride ligands while the rest of the molecule is not altered; alternatively, activation may occur through gold(III) reduction. All compounds react eagerly with the model protein cyt c leading to extensive protein metalation. ESI MS experiments revealed details of gold-cyt c interactions and allowed us to establish the nature of protein bound metal containing fragments. The different behavior displayed by I and II compared to III and IV is highlighted. Remarkable cytotoxic properties, against the reference human ovarian carcinoma cell lines A2780/S and A2780/R were disclosed for all tested compounds with IC(50) values ranging from 1.43 to 6.18 μM in the sensitive cell line and from 1.59 to 10.86 μM in the resistant one. The common ability of these compounds to overcome cisplatin resistance is highlighted. The obtained results are thoroughly discussed in the frame of current knowledge on cytotoxic gold compounds.

A series of new gold(I) and gold(III) complexes based on the saccharinate (sac) ligand, namely M[Au( sac ) 2 ] (with M being Na + , K + or NH 4 + ), [(PTA)Au( sac )], K[Au( sac ) 3 Cl] and Na[Au( sac ) 4 ], were synthesized and characterized, and some aspects of their biological profile investigated. Spectrophotometric analysis revealed that these gold compounds, upon dissolution in aqueous media, at physiological pH, manifest a rather favourable balance between stability and reactivity. Their reactions with the model proteins cytochrome c and lysozyme were monitored by mass spectrometry to predict their likely interactions with protein targets. In the case of disaccharinato gold(I) complexes, cytochrome c adducts bearing four coordinated gold(I) ions were preferentially formed in high yield. In contrast, [(PTA)Au(sac)] (PTA = 1,3,5-triaza-7-phosphaadamantane) turned out to be poorly effective, only producing a mono-metalated adduct in very low amount. In turn, the gold(III) saccharinate derivatives were less reactive than their gold(I) analogues: K[Au(sac) 3 Cl] and Na[Au(sac) 4 ] caused moderate protein metalation, again with evidence of formation of tetragold adducts. Finally, the above mentioned gold compounds were challenged against the reference human tumor cell line A2780S and its cisplatin resistant subline A2780R and their respective cytotoxic profiles determined. [(PTA)Au( sac )] turned out to be highly cytotoxic whereas moderate cytotoxicities were observed for the gold(III) complexes and only modest activities for disaccharinato gold(I) complexes. The implications of these results are thoroughly discussed in the light of current knowledge on gold based drugs.

The reaction of aqueous cis-[Pt(NH(3))(2)(H(2)O)(2)](NO(3))(2) with Na(+)HMEL(-) (H(2)MEL, meloxicam, 4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide), and Na(+)HISO(-)(H(2)ISO, isoxicam, 4-hydroxy-2-methyl-N-(5-methylisoxazol-3-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide) at pH 7 produced micro-crystalline cis-[Pt(NH(3))(2)((N) under bar (1)'-HMEL)(2)], 5 and cis-[Pt(NH(3))(2)((N) under bar (1)'-HISO)(2)], 6. The X-ray diffraction structure of 5 shows two HMEL(-) anions donating through the thiazole nitrogen atoms and adopting a head-to-tail (HT) conformation. The (1)H NMR spectrum for 5 from DMSO-d(6) shows inertness of the complex up to at least 24 h. Delivery studies for 5 and 6 from vinyl hydrogel based on L-phenylalanine (pH 6.5, 25 degrees C) show that concentrations of complexes ranging between 2.5 and 5 mu M can be reached after a day. Compounds 5 and 6 show strong anti-proliferative effects on CH1 cells (ovarian carcinoma, human) in vitro. IC(50) values being 0.60 and 0.37 mu M, respectively (0.16 mu M for reference, cis-diamminodichloridoplatinum(II), cisplatin). ESI-MS measurements clearly documented that both 5 and 6 form adducts with the three model proteins ubiquitin (UBI), cytochrome c (CYT C) and superoxide dismutase (SOD), the HISO(-) complex being significantly more effective than the HMEL(-) one. Density functional methods help in finding rationale for the easiest dissociation of Pt-H(2)ISO/HISO bonds when compared to the Pt-(N) under bar (1)'-H(2)MEL/(N) under bar (1)'-HMEL linkages. (C) 2010 Elsevier Inc. All rights reserved

A novel "Keppler type" ruthenium(III) compound trans-[bis(2-amino 5-methylthiazole)tetrachlororuthenate(III)] 1, of potential interest as an anticancer agent, was designed, synthesized, and characterized. Its interactions with various proteins were analyzed, including the selenoenzyme thioredoxin reductase, an emerging target for anticancer metallodrugs. The selective inhibition of the cytosolic form of this selenoenzyme was documented, this being the first report of significant thioredoxin reductase inhibition by a ruthenium compound.

The investigation of cis-PtI2(NH3)2, the diiodido analogue of cisplatin (cisPtI2 hereafter), has been unjustly overlooked so far mainly because of old claims of pharmacological inactivity. Some recent - but still fragmentary - findings prompted us to reconsider more systematically the chemical and biological profile of cisPtI2 in comparison with cisplatin. Its solution behaviour, interactions with DNA and cytotoxic properties versus selected cancer cell lines were thus extensively analysed through a variety of biophysical and computational methods. Notably, we found that cisPtI2 is highly cytotoxic in vitro toward a few solid tumour cell lines and that its DNA platination pattern closely reproduces that of cisplatin; cisPtI2 is also shown to completely overcome resistance to cisplatin in a platinum resistant cancer cell line. The differences in the biological actions of these two Pt complexes are most likely related to slight but meaningful differences in their solution behaviour and reactivity. Overall, a very encouraging and unexpected pharmacological profile emerges for cisPtI2 with relevant implications both in terms of mechanistic knowledge and of prospective clinical application. An ab initio DFT study is also included to support the interpretation of the solution behaviour of cisPtI2 under physiological and slightly acidic pH conditions, TM and LM acknowledge Beneficentia Stiftung (Vaduz), COST Action CM1105 and AIRC (IG-16049) for generous financial support. JK and VB acknowledge the support from the Czech Science Foundation (Grant 14-21053S)

A series of six dinuclear gold(III) oxo complexes with bipyridyl ligands, of general formula [Au2(N,N)2(mu-O)2][PF6]2 (Auoxo1-Auoxo6) [where N,N = 2,2'-bipyridine (1), 4,4'-di-tert-butyl- (2), 6-methyl- (3), 6-neopentyl- (4), 6-(2,6-dimethylphenyl)- (5), 6,6'-dimethyl-2,2'-bipyridine (6)], were investigated as potential cytotoxic and anticancer agents, and their antiproliferative properties were evaluated in vitro toward the reference A2780 human ovarian carcinoma cell line. While five compounds manifested moderate cytotoxic properties (with IC50 approximately 10-30 microM), the sixth one (Auoxo6), turned out to be approximately 5-15 times more active against both cell lines and will merit further pharmacological studies. The interactions of Auoxo1 and Auoxo6 with a few model proteins (serum albumin, cytochrome c, ubiquitin) and with calf thymus DNA were analyzed in detail by various spectroscopic methods. Both tested compounds manifested a high and peculiar reactivity toward the mentioned model proteins; specific differences were detected in their reactivity with DNA. The mechanistic implications of these results are discussed.

The cellular alterations produced in cisplatin-resistant A2780 ovarian cancer cells (A2780/R) upon treatment with the cytotoxic organogold(III) complex Aubipyc were investigated in depth through a classical proteomic approach. We observed that A2780/R cell exposure to a cytotoxic concentration of Aubipyc for 24 hours results in a conspicuous number of alterations at the protein level that were carefully examined. Notably, we observed that several affected proteins belong to the glucose metabolism system further supporting the idea that the cytotoxic effects of Aubipyc in A2780/R cells are mostly mediated by an impairment of glucose metabolism in excellent agreement with previous observations on the parent cisplatin-sensitive cell line.

The so called “copper trafficking system” in mammalian cells is primarily devoted to the regulation of copper transport and homeostasis. This system, now well characterized, consists of a few strictly interconnected proteins that assist copper entrance inside cells and then promote metal transfer and delivery to essential copper-dependent cellular proteins (Boal and Rosenzweig 2009a; Banci et al., Mol Life Sci 67:2563–2589, 2010). Yet, the “copper trafficking system” may also facilitate the entrance inside cells of non-physiological metal species such as clinically established platinum drugs. ESI and MALDI MS methods are exploited here to characterize the interactions occurring between the experimental anticancer organogold(III) drug, Aubipyc, and the copper chaperone Atox1, a key protein of the copper trafficking system. The nature of the adducts that are formed when reacting Aubipyc with Atox1 is elucidated in detail. Characterization of the Aubipyc/Atox1 system is further supported by circular dichroism experiments. Binding competitions with mercury and bismuth ions were also explored. The relevance and the biological implications of the present results are discussed.

Cisplatin and its analogues are first-line chemotherapeutic agents for the treatment of numerous human cancers. A major inconvenience in their clinical use is their strong tendency to link to sulfur compounds, especially in kidney, ultimately leading to severe nephrotoxicity. To overcome this drawback we prepared a variety of platinum complexes with sulfur ligands and analyzed their biological profiles. Here, a series of six platinum(II) compounds bearing a conserved O,S binding moiety have been synthesized and characterized as experimental anticancer agents. The six compounds differ in the nature of the O,S bidentate β-hydroxydithiocinnamic alkyl ester ligand where both the substituents on the aromatic ring and the length of the alkyl chain may be varied. The two remaining coordination positions at the square-planar platinum(II) center are occupied by a chloride ion and a DMSO molecule. These novel platinum compounds showed an acceptable solubility profile in mixed DMSO-buffer solutions and an appreciable stability at physiological pH as judged from analysis of their time-course UV-visible absorption spectra. Their anti-proliferative and pro-apoptotic activities were tested against the cisplatin-resistant lung cancer cell line A549. Assays revealed significant effects of the sample drugs at low concentrations (in the μmolar range); initial structure-activity-relationships are proposed. The activity of the apoptosis-promoting protein caspase 3/7 was determined; results proved that these novel platinum compounds, under the chosen experimental conditions, preferentially induce apoptosis over necrosis. Reactions with the model proteins cytochrome c, lysozyme and albumin were studied by ESI MS and ICP-OES to gain preliminary mechanistic information. The tested compounds turned out to metalate the mentioned proteins to a large extent. In view of the obtained results these novel platinum complexes qualify themselves as promising cytotoxic agents and merit, in our opinion, a deeper pharmacological evaluation as prospective anticancer agents.

Six structurally diverse cytotoxic gold compounds are reported to cause profound and differential inhibition of the three main catalytic activities of purified 20S proteasome whilst auranofin , an established gold(I) drug in clinical use, is nearly ineffective. In particular, the gold(I) complex [( pbiH ) Au ( PPh 3 )] PF 6 , turns out to be the most potent inhibitor of all three enzyme activities with sub-micromolar IC 50 values. The present results further support the view that proteasome inhibition may play a major – yet not exclusive – role in the cytotoxic actions of gold based anticancer agents.

Keppler-type ruthenium (III) complexes exhibit promising antitumor properties. We report here a study of 2-aminothiazolium[trans-tetrachlorobis(2-aminothiazole)ruthenate(III)], both in the solid state and in solution. The crystal structure has been solved and found to exhibit classical features. Important solvatochromic effects were revealed. Notably, we observed that introduction of an amino group in position 2 greatly accelerates chloride hydrolysis compared to the thiazole analogue; this latter finding may be of interest for a fine-tuning of the reactivity of these novel metallodrugs.

DIGE (difference in gel electrophoresis) proteomics is exploited here to gain insight into the molecular mechanisms of two established ruthenium-based antimetastatic agents, namely trans-[tetrachloro (DMSO) (imidazole)ruthenate(III)] (NAMI-A) and [Ru(eta(6)-toluene)Cl-2(PTA)] (RAPTA-T), where PTA is 1,3,5-triaza-7-phosphaadamantane. Following 24 h exposure of A2780/S human ovarian carcinoma cells to pharmacologically relevant concentrations of either ruthenium compound, 2D-DIGE proteomic analysis evidenced only few differentially expressed proteins with respect to controls. Successive mass spectrometry measurements, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) or LC-ESI/MS-MS (liquid chromatography-electrospray ionization/multi-stage mass spectrometry), allowed identification of most altered protein spots, some of which were associated to perturbations in specific cellular functions. Direct insight into the cellular effects of the investigated metallodrugs is thus achieved. Notably, the patterns of protein alterations induced by NAMI-A and RAPTA-T are quite similar to each other while being deeply different from those of cisplatin. To the best of our knowledge this is the first proteomic study on human cancer cells investigating responses to antimetastatic ruthenium drugs. The key role of new "omic" approaches for deciphering the elusive and complex biochemical mechanisms through which anticancer metallodrugs produce their pharmacological effects is further documented. (C) 2012 Elsevier Inc. All rights reserved.

Thioredoxin reductase (TrxR) is overexpressed in cancer cells and is therefore a putative cancer target. Inhibition of this enzyme is considered an important strategy for the development of new chemotherapeutic agents with a specific mechanism of action. Organotin compounds have been described as experimental antitumor agents, yet their mechanism of action remains largely unknown. Based on the outcome of a virtual screening study, various di- and tri-n-butyltin(IV) carboxylates were synthesized, and their biological properties were evaluated. All synthesized compounds were able to inhibit TrxR selectively within the micromolar range and showed potent antitumor activity against HT-29 and MCF-7 cancer cell lines. Moreover, tin(IV) organometallics were found to strongly induce apoptosis in the BJAB lymphoma cell line. Mass spectrometry and atomic absorption spectroscopy experiments revealed metal binding to proteins, and efficient cellular uptake was observed using a di-n-butyltin(IV) complex as an example.

A variety of gold(III) and gold(I) derivatives of 2-(2'-pyridyl)benzimidazole (pbiH) were synthesized and fully characterized and their antiproliferative properties evaluated in a representative ovarian cancer cell line. The complexes include the mononuclear species [(pbi)AuX(2)] (X = Cl, 1; OAc, 2), [(pbiH)AuCl] (3), [(pbiH)Au(PPh(3))][PF(6)] (4-PF(6)), and [(pbi)Au(L)] (L = PPh(3), 5; TPA, 6), and the binuclear gold(I)/gold(I) and gold(I)/gold(III) derivatives [(PPh(3))(2)Au(2)(μ(2)-pbi)][PF(6)] (10-PF(6)), [ClAu(μ(3)-pbi)AuCl(2)] (7),and [(PPh(3))Au(μ(3)-pbi)AuX(2)][PF(6)] (X = Cl, 8-PF(6); OAc, 9-PF(6)). The molecular structures of 6, 7, and 10-PF(6) were determined by X-ray diffraction analysis. The chemical behavior of these compounds in solution was analyzed both by cyclic voltammetry in DMF and absorption UV-vis spectroscopy in an aqueous buffer. Overall, the stability of these gold compounds was found to be acceptable for the cellular studies. For all complexes, relevant antiproliferative activities in vitro were documented against A2780 human ovarian carcinoma cells, either resistant or sensitive to cisplatin, with IC(50) values falling in the low micromolar or even in the nanomolar range. The investigated gold compounds were found to overcome resistance to cisplatin to a large degree. Results are interpreted and discussed in the frame of current knowledge on cytotoxic and antitumor gold compounds.

Six diiodido-diamine platinum(II) complexes, either cis or trans configured, were prepared, differing only in the nature of the amine ligand (isopropylamine, dimethylamine, or methylamine), and their antiproliferative properties were evaluated against a panel of human tumor cell lines. Both series of complexes manifested pronounced cytotoxic effects, with the trans isomers being, generally, more effective than their cis counterparts. Cell cycle analysis revealed different modes of action for these new Pt(II) complexes with respect to cisplatin. The reactivity of these platinum compounds with a number of biomolecules, including cytochrome c, two sulfur containing modified amino acids, 9-ethylguanine, and a single strand oligonucleotide, was analyzed in depth by mass spectrometry and NMR spectroscopy. Interestingly, significant differences in the reactivity of the investigated compounds toward the various model biomolecules were observed: in particular we observed that trans complexes preferentially release their iodide ligands upon biomolecule binding, while the cis isomers may release the amine ligands with retention of iodides. Such differences in reactivity may have important mechanistic implications and a relevant impact on the respective pharmacological profiles.

Protein metalation processes are crucial for the mechanism of action of several anticancer metallodrugs and warrant deeper characterisation. We have explored the reactions of three cytotoxic gold(III) compounds-namely [(bipy(2Me))(2)Au(2)(μ-O)(2)][PF(6)](2) (where bipy(2Me) is 6,6'-dimethyl-2,2'-bipyridine) (Auoxo6), [(phen(2Me))(2)Au(2)(μ-O)(2)][PF(6)](2) (where phen(2Me) is 2,9-dimethyl-1,10-phenanthroline) (Au(2)phen) and [(bipy(dmb)-H)Au(OH)][PF(6)] [where bipy(dmb)-H is deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine] (Aubipyc)-with two representative model proteins, i.e. horse heart cytochrome c and hen egg white lysozyme, through UV-visible absorption spectroscopy and electrospray ionisation mass spectrometry (ESI MS) to characterise the inherent protein metalation processes. Notably, Auoxo6 and Au(2)phen produced stable protein adducts where one or more "naked" gold(I) ions are protein-coordinated; very characteristic is the case of cytochrome c, which upon reaction with Auoxo6 or Au(2)phen preferentially forms "tetragold" adducts with four protein-bound gold(I) ions. In turn, Aubipyc afforded monometalated protein adducts where the structural core of the gold(III) centre and its +3 oxidation state are conserved. Auranofin yielded protein derivatives containing the intact auranofin molecule. Additional studies were performed to assess the role played by a reducing environment in protein metalation. Overall, the approach adopted provides detailed insight into the formation of metallodrug-protein derivatives and permits trends, peculiarities and mechanistic details of the underlying processes to be highlighted. In this respect, electrospray ionisation mass spectrometry is a very straightforward and informative research tool. The protein metalation processes investigated critically depend on the nature of both the metal compound and the interacting protein and also on the solution conditions used; thus, predicting with accuracy the nature and the amounts of the adducts formed for a given metallodrug-protein pair is currently extremely difficult.

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.

Reactions of cytotoxic platinum drugs with proteins are attracting growing attention for their relevant biological implications. We report here on the reactions of two cis-diphosphane platinum(II) dichlorides (namely cis-bis(trimethylphosphane) platinum(II) dichloride and cis-bis(triethylphosphane) platinum(II) dichloride) with horse heart cytochrome c (cyt c) monitored through advanced ESI MS methods coupled to enzymatic digestion. A remarkable selectivity in terms of adduct stoichiometry is highlighted and the specific metal binding sites are localised on the protein surface.

Gold compounds form an interesting class of antiproliferative agents of potential pharmacological use in cancer treatment. Indeed, a number of gold compounds, either gold(III) or gold(I), were recently described and characterised that manifested remarkable cytotoxic properties in vitro against cultured cancer cells; for some of them encouraging in vivo results were also reported toward a few relevant animal models of cancer. The molecular mechanisms through which gold compounds exert their biological effects are still largely unknown and the subject of intense investigations. Recent studies point out that the modes of action of cytotoxic gold compounds are essentially DNA-independent and cisplatin-unrelated, relying -most likely- on gold interactions with a variety of protein targets. Notably, a few cellular proteins playing relevant functional roles were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses need adequate validation. The state of the art of this research area and the perspectives for future studies are herein critically analysed and discussed.

Since the discovery of cisplatin and its introduction in the clinics, metal compounds have been intensely investigated in view of their possible application in cancer therapy. In this frame, a deeper understanding of their mode of action, still rather obscure, might turn crucial for the design and the obtainment of new and better anticancer agents. Due to the extreme complexity of the biological systems, it is now widely accepted that innovative and information-rich methods are absolutely needed to afford such a goal. Recently, both proteomic and metallomic strategies were successfully implemented for the elucidation of specific mechanistic features of anticancer metallodrugs within an innovative “Systems Biology” perspective. Particular attention was paid to the following issues: i) proteomic studies of the molecular basis of platinum resistance; ii) proteomic analysis of cellular responses to cytotoxic metallodrugs; iii) metallomic studies of the transformation and fate of metallodrugs in cellular systems. Notably, those pioneering studies, that are reviewed here, allowed a significant progress in the understanding of the molecular mechanisms of metal based drugs at the cellular level. A further extension of those studies and a closer integration of proteomic and metallomic strategies and technologies might realistically lead to rapid and significant advancements in the mechanistic knowledge of anticancer metallodrugs.

Gold compounds are a class of metallodrugs with great potential for cancer treatment. During the last two decades, a large variety of gold(I) and gold(III) compounds are reported to possess relevant antiproliferative properties in vitro against selected human tumor cell lines, qualifying themselves as excellent candidates for further pharmacological evaluation. The unique chemical properties of the gold center confer very interesting and innovative pharmacological profiles to gold-based metallodrugs. The primary goal of this review is to define the state of the art of preclinical studies on anticancer gold compounds, carried out either in vitro or in vivo. The available investigations of anticancer gold compounds are analyzed in detail, and particular attention is devoted to underlying molecular mechanisms. Notably, a few biophysical studies reveal that the interactions of cytotoxic gold compounds with DNA are generally far weaker than those of platinum drugs, implying the occurrence of a substantially different mode of action. A variety of alternative mechanisms were thus proposed, of which those involving either direct mitochondrial damage or proteasome inhibition or modulation of specific kinases are now highly credited. The overall perspectives on the development of gold compounds as effective anticancer drugs with an innovative mechanism of action are critically discussed on the basis of the available experimental evidence.

8 páginas, 6 figuras, 2 tablas -- PAGS nros. 9139-9146, A novel trans-platinum(II) complex bearing one dimethylamine (dma) and one methylamine (ma) ligand, namely trans-[PtCl(2)(dma)(ma)], recently synthesised and characterised in our laboratory, displayed relevant antiproliferative properties in vitro, being more active than the parent complex, trans-[PtCl(2)(dma)(ipa)], which has isopropylamine (ipa) in place of methylamine. We have analysed comparatively the solution behaviour of these two complexes under various experimental conditions, and investigated their reactivity with horse heart cytochrome c by mass spectrometry, inductively coupled plasma-optical emission spectroscopy (ICP-OES), 2D [(1)H,(15)N],[(1)H,(13)C] HSQC and [(1)H,(1)H] NOESY NMR. Some important changes that occurred in the [(1)H,(13)C] HSQC NMR spectrum of cytochrome c treated with trans-[PtCl(2)(dma)(ma)] in water, after two days' incubation, most probably arose from direct platinum coordination to the protein side chain; this was proved conclusively by [(1)H,(1)H] NOESY NMR and [(1)H,(15)N] HSQC NMR measurements. Met65 was identified as the primary Pt binding site on cytochrome c. Electrospray mass spectrometry (ESIMS) results provided evidence for extensive platinum-protein adduct formation. A fragment of the [Pt(amine)(amine')] type was established to be primarily responsible for protein metalation. ICP-OES analysis revealed that these trans-platinum(II) complexes bind preferentially to the serum proteins albumin and transferrin rather than to calf thymus DNA. Pt binding to DNA was found to be far lower than in the case of cisplatin. The implications of the results for the mechanism of action of novel cytotoxic trans-platinum complexes are discussed, We would like to thank the International cooperation program: HI2007-0153 and the COST D39 Action for financial support, in particular WG6. C.N.R., A.G.Q. and L.C. thank the Spanish Comisión Interministerial de Ciencia y Tecnología (CICYT-SAF-2006-03296) for funding. A.C. thanks the Swiss National Science Foundation for financial support (Ambizione project no. PZ00P2_121933)

There is considerable interest today for the reactions of anticancer metallodrugs with proteins as these interactions might feature processes that are crucial for the biodistribution, the toxicity and even the mechanism of action of this important group of anticancer agents. We survey here the results of research activities carried out in our "Laboratory of Metals in Medicine" (Department of Chemistry, University of Florence) during the last three years, concerning the molecular characterisation of adducts formed between platinum, ruthenium and gold metallodrugs and a few model proteins. Valuable structural and functional information on these adducts could be derived from several biophysical studies mainly relying on the application of X-ray diffraction and ESI MS techniques. The value and the limitations of both approaches are outlined through a number of examples. Remarkably, the structural and functional information achieved on the respective metallodrug-protein adducts allowed us to identify some general trends in the reactivity of anticancer metallodrugs with protein targets.

A series of ruthenium(II)-arene (RAPTA) compounds were evaluated for their ability to inhibit thioredoxin reductase (either cytosolic or mitochondrial) and cathepsin B, two possible targets for anticancer metallodrugs. In general, inhibition of the thioredoxin reductases was lower than that of cathepsin B, although selected compounds were excellent inhibitors of both classes of enzymes in comparison to other metal-based drugs. Some initial structure-activity relationships could be established. On the basis of the obtained data, different mechanisms of binding/inhibition appear to be operative; remarkably the selectivity of the ruthenium compounds toward solid metastatic tumors also correlates to the observed trends. Notably, docking studies of the interactions of representative RAPTA compounds with cathepsin B were performed that provided realistic structures for the resulting protein-metallodrug adducts. Good agreement was generally found between the inhibiting potency of the RAPTA compounds and the computed stability of the corresponding cat B/RAPTA adducts.

The interactions of anticancer metallodrugs with proteins are attracting a growing interest in the current literature because of their relevant pharmacological and toxicological consequences. To understand in more depth the nature of those interactions, we have investigated the reactions of four anticancer platinum(II) iminoether complexes, namely, trans- and cis-EE (trans- and cis-[PtCl2{(E)-HN=C(OCH3)CH3}2], respectively) and trans- and cis-Z (trans- and cis-[PtCl2(NH3){(Z)-HN=C(OCH3)CH3}], respectively), with horse heart cytochrome c (cyt c). Our investigation was performed using mainly electrospray ionization mass spectrometry (ESI MS) but was also supported by NMR, inductively coupled plasma optical emission spectroscopy (ICP OES), and absorption electronic spectroscopy. ESI MS spectra clearly revealed the formation of a variety of platinum-protein adducts predominantly corresponding to monoplatinated cyt c species. From a careful analysis of the major ESI MS peaks, specific information on the nature of the protein-bound metallic fragments and on the underlying metallodrug-cyt c reactions was gained for the various cases. We found that trans-EE produces a major cyt c adduct (12 667 Da) that is different from that produced by either cis-EE or by trans-Z and cis-Z (12 626 Da). In particular, occurrence of extensive hydrolysis/aminolysis (the latter fostered by ammonium carbonate buffer) of the iminoether ligands and formation of the corresponding amides/amidines has been unambiguously documented. The reactivity of the iminoether ligands is greatly enhanced by the presence of cyt c as inferred from comparative NMR solution studies. Additional ESI MS measurements recorded on enzymatically cleaved samples of platinated cyt c adducts, together with NMR investigation of the cyt c/trans-EE adduct, strongly suggest that protein platination primarily occurs at Met 65. The biological and pharmacological implications of the described protein platination processes are discussed.

The interactions with protein targets of the ruthenium(III) complex imidazolium trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, an effective anticancer and antimetastatic agent now in clinical trials, deserve great attention as they are believed to be at the basis of the mechanism of action of this innovative molecule. Here, we report on the reactions of NAMI-A with two well-known model proteins, namely, hen egg white lysozyme and horse heart cytochrome c; these reactions were investigated by a variety of physicochemical methods, including optical spectroscopy, (1)H NMR and electrospray ionization mass spectrometry. The combined use of the analytical techniques mentioned resulted in a rather exhaustive description of the NAMI-A-protein interactions; in particular, the formation of fairly stable metal-protein adducts was clearly documented and the nature of the resulting protein-bound metallic fragments ascertained in most cases. Notably, greatly different patterns of interaction were found to be operative for NAMI-A toward these two proteins. The biological implications of the present findings are discussed.

Electrospray ionization mass spectrometry allows a rapid characterisation of the adducts formed between three novel anticancer ruthenium(II)-arene PTA compounds and horse heart cytochrome c or hen egg white lysozyme. Specific information on the nature of the protein-bound metallic fragments and the extent of protein metallation was readily obtained.

The effects of a few cytotoxic organogold(III) compounds on ovarian A2780 human cancer cells were investigated in comparison to cisplatin and oxaliplatin. The tested compounds produced significant antiproliferative effects and promoted apoptosis to a greater extent than platinum drugs while causing only modest cell cycle modifications. The mechanistic implications of these findings are discussed: mitochondrial pathways are proposed to be directly involved in the apoptotic process in relation to selective inhibition of thioredoxin reductase.

The solution behaviour of some novel organogold(III) compounds was investigated, and their cytotoxic properties evaluated against a few human tumour cell lines (A2780/S, A2780/R, MCF7, HT29 and A549). Specifically, the following compounds were considered: [Au(bipy dmb -H)(2,6-xylidine-H)][PF 6 ] (AuXyl) and [Au(bipy dmb -H)( p -toluidine-H)][PF 6 ] (AuTol) (in which bipy dmb = 6-(1,1-dimethylbenzyl)-2,2′-bipyridine), [Au(py dmb -H)(AcO) 2 ] (AuPyAcO) (in which py dmb = 2-(1,1-dimethylbenzyl)-pyridine) and [Au(pz Ph -H)Cl 3 ]K (AuPzCl) (in which pz Ph = 1-phenylpyrazole). The solution chemistry of these compounds, under physiological-like conditions, was investigated through UV–vis absorption and 1 H NMR spectroscopies. Significant cytotoxic effects in vitro were observed in selected cases.

The interactions between a few representative gold-based drugs and hen egg white lysozyme were studied by X-ray crystallography. High resolution crystal structures solved for three metallodrug-protein adducts provide valuable insight into the molecular mechanism of these promising metal compounds and the inherent protein metalation processes.

Two dinuclear oxo-bridged organogold(III) compounds, namely [(N,N,C)(2)Au(2)(μ-O)][PF(6)](2) (with N,N,CH = 6-(1-methylbenzyl)-2,2'-bipyridine, Au(2)O1; or 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, Au(2)O2), were previously prepared and characterised. Their solution chemistry under physiological-like conditions has been investigated here as well as their in vitro antiproliferative properties. Notably, these compounds reveal a marked redox stability even in the presence of effective biological reductants such as ascorbic acid and glutathione. The two dinuclear gold(iii) compounds were evaluated for cytotoxic actions against a representative panel of 12 human tumor cell lines, in comparison to respective mononuclear parent compounds [(N,N,C)AuOH][PF(6)], and appreciable biological activity could be highlighted. The reactions of Au(2)O1 and Au(2)O2 with a few model proteins were studied and the ability to form metallodrug-protein adducts monitored through ESI MS methods. Typical adducts were identified where the protein is associated to monometallic gold fragments; in these adducts gold remains in the oxidation state +3 and conserves its organic ligand. A direct comparison of the biological profiles of these binuclear organogold(III) compounds with those previously reported for a series of dinuclear oxo-bridged complexes [(N,N)(2)Au(2)(μ-O)(2)][PF(6)](2) (N,N = 6(6')-substituted 2,2'-bipyridines) named Auoxo's was carried out. It emerges that the greater cytotoxicity of the latter is mainly due to the greater oxidising power of their gold(III) centres and to propensity to generate gold(i) species; in contrast, the here described bimetallic organogold(III) complexes manifest a far higher redox stability in the biological milieu coupled to lower, but still significant, antiproliferative properties. Different molecular mechanisms are thus hypothesised for these two classes of dinuclear gold(III) agents.

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 effects 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 effects 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 effects. 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 formation and the nature of gold-protein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectrophotometric studies of appropriate model systems as it is exemplified 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 specifically assessed. Perspectives for the future of this topics are briefly outlined.

Carboplatin (CPT), today the most important platinum(II) anticancer drug, manifests an extreme kinetic inertness, in vitro, at physiological pH; the actual mechanisms for its activation inside cells are still poorly understood. We show here that horse heart cytochrome c reacts with CPT, leading to the formation of stable platinum/protein adducts. The two major CPT-cytochrome c species resulting from the aforementioned reaction were characterised by electrospray ionisation mass spectrometry (ESI-MS). Notably, both these adducts have the ability to react with guanosine 5'-monophosphate (5'-GMP), giving rise to the respective cytochrome c-CPT-5'-GMP ternary complexes. Additional ESI-MS measurements on enzymatically cleaved cytochrome c adducts suggest that protein platination probably occurs at Met65. The mechanistic implications of these findings are discussed.

Heavy metals rock! The reactivity of the antimetastatic metallodrug NAMI-A with human carbonic anhydrase II (hCAII) has been investigated using a number of techniques, including X-ray crystallography. The characterization of the interactions between NAMI-A and hCAII provides valuable information on the molecular mechanisms responsible for the activity of this promising anticancer agent.