Diruthenium Paddlewheel Complexes Attacking Proteins: Axial versus Equatorial Coordination.

Metallodrugs are an important group of medicinal agents used for the treatment of various diseases ranging from cancers to viral, bacterial, and parasitic diseases. Their distinctive features include the availability of a metal centre, redox activity, as well as the ability to multitarget. Diruthenium paddlewheel complexes are an intensely developing group of metal scaffolds, which can securely coordinate bidentate xenobiotics and transport them to target tissues, releasing them by means of substitution reactions with biomolecular nucleophiles. It is of the utmost importance to gain a complete comprehension of which chemical reactions happen with them in physiological milieu to design novel drugs based on these bimetallic scaffolds. This review presents the data obtained in experiments and calculations, which clarify the chemistry these complexes undergo once administered in the proteic environment. This study demonstrates how diruthenium paddlewheel complexes may indeed embody a new paradigm in the design of metal-based drugs of dual-action by presenting and discussing the protein metalation by these complexes.

Evaluation of Auranofin Loading within Ferritin Nanocages.

Auranofin (AF), a gold(I) compound that is currently used for the treatment of rheumatoid arthritis and is in clinical trials for its promising anticancer activity, was encapsulated within the human H-chain and the horse spleen ferritin nanocages using the alkaline disassembly/reassembly protocol. The aim of the work was to highlight possible differences in their drug loading capacity and efficacy. The drug-loaded ferritins were characterized via UV-vis absorption spectroscopy and inductively coupled plasma-atomic emission spectroscopy to assess AF encapsulation and to define the exact amount of gold atoms trapped in the Ft cavity. The crystal structures allowed us to define the nature of AF interaction with both ferritins and to identify the gold binding sites. Moreover, the biological characterization let us to obtain preliminary information on the cytotoxic effect of AF when bound to the human H-chain.

Protein-Based Delivery Systems for Anticancer Metallodrugs: Structure and Biological Activity of the Oxaliplatin/ß-Lactoglobulin Adduct.

ß-lactoglobulin is the major component of whey. Here, the adduct formed upon the reaction of the protein with oxaliplatin (OXA) has been prepared, structurally characterized by X-ray crystallography and electrospray ionization-mass spectrometry, and evaluated as a cytotoxic agent. The data demonstrate that OXA rapidly binds ß-lactoglobulin via coordination with a Met7 side chain upon release of the oxalate ligand. The adduct is significantly more cytotoxic than the free drug and induces apoptosis in cancer cells. Overall, our results suggest that metallodrug/ß-lactoglobulin adducts can be used as anticancer agents and that the protein can be used as a metallodrug delivery system.

Arsenoplatin-Ferritin Nanocage: Structure and Cytotoxicity.

Arsenoplatin-1 (AP-1), the prototype of a novel class of metallodrugs containing a PtAs(OH)2 core, was encapsulated within the apoferritin (AFt) nanocage. UV-Vis absorption spectroscopy and inductively coupled plasma-atomic emission spectroscopy measurements confirmed metallodrug encapsulation and allowed us to determine the average amount of AP-1 trapped inside the cage. The X-ray structure of AP-1-encapsulated AFt was solved at 1.50 Å. Diffraction data revealed that an AP-1 fragment coordinates the side chain of a His residue. The biological activity of AP-1-loaded AFt was comparatively tested on a few representative cancer and non-cancer cell lines. Even though the presence of the cage reduces the overall cytotoxicity of AP-1, it improves its selectivity towards cancer cells.

Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme.

The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh2(µ-O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh2(µ-O2CCH3)4] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side chains of Arg14 and His15. Dimeric Rh-Rh units with Rh-Rh distances between 2.3 and 2.5 Å are bound to the side chains of Asp18, Asp101, Asn93, and Lys96, while a dirhodium unit with a Rh-Rh distance of 3.2-3.4 Å binds the C-terminal carboxylate and the side chain of Lys13 at the interface between two symmetry-related molecules. An additional monometallic fragment binds the side chain of Lys33. These data, which are supported by replicated structural determinations, shed light on the reactivity of dirhodium tetracarboxylates with proteins, providing useful information for the design of new Rh-containing biomaterials with an array of potential applications in the field of catalysis or of medicinal chemistry and valuable insight into the mechanism of action of these potential anticancer agents.

Interaction of Platinum-based Drugs with Proteins: An Overview of Representative Crystallographic Studies.

Pt-based drugs are widely used in clinics for the treatment of cancer. The mechanism of action of these molecules relies on their interaction with DNA. However, the recognition of these metal compounds by proteins plays an important role in defining pharmacokinetics, side effects and their overall pharmacological profiles. Single crystal X-ray diffraction studies provided important information on the molecular mechanisms at the basis of this process. Here, the molecular structures of representative adducts obtained upon reaction with proteins of selected Pt-based drugs, including cisplatin, carboplatin and oxaliplatin, are briefly described and comparatively examined. Data indicate that metal ligands play a significant role in driving the reaction of Pt compounds with proteins; non-covalent interactions that occur in the early steps of Pt compound/protein recognition process play a crucial role in defining the structure of the final Pt-protein adduct. In the metallated protein structures, Pt centers coordinate few protein side chains, such as His, Met, Cys, Asp, Glu and Lys residues upon releasing labile ligands.

Mechanistic Insights Into the Anticancer Properties of the Auranofin Analog Au(PEt3)I: A Theoretical and Experimental Study.

Au(PEt3)I (AF-I hereafter), the iodide analog of the FDA-approved drug auranofin (AF hereafter), is a promising anticancer agent that produces its pharmacological effects through interaction with non-genomic targets such as the thioredoxin reductase system. AF-I is endowed with a very favorable biochemical profile showing potent in vitro cytotoxic activity against several cancer types including ovarian and colorectal cancer. Remarkably, in a recent publication, some of us reported that AF-I induces an almost complete and rapid remission in an orthotopic in vivo mouse model of ovarian cancer. The cytotoxic potency does not bring about highly severe side effects, making AF-I very well-tolerated even for higher doses, even more so than the pharmacologically active ones. All these promising features led us to expand our studies on the mechanistic aspects underlying the antitumor activity of AF-I. We report here on an integrated experimental and theoretical study on the reactivity of AF-I, in comparison with auranofin, toward relevant aminoacidic residues or their molecular models. Results point out that the replacement of the thiosugar moiety with iodide significantly affects the overall reactivity toward the amino acid residues histidine, cysteine, methionine, and selenocysteine. Altogether, the obtained results contribute to shed light into the enhanced antitumoral activity of AF-I compared with AF.

On the Different Mode of Action of Au(I)/Ag(I)-NHC Bis-Anthracenyl Complexes Towards Selected Target Biomolecules.

Gold and silver N-heterocyclic carbenes (NHCs) are emerging for therapeutic applications. Multiple techniques are here used to unveil the mechanistic details of the binding to different biosubstrates of bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) silver chloride [Ag(EIA)2]Cl and bis(1-(anthracen-9-ylmethyl)-3-ethylimidazol-2-ylidene) gold chloride [Au(EIA)2]Cl. As the biosubstrates, we tested natural double-stranded DNA, synthetic RNA polynucleotides (single-poly(A), double-poly(A)poly(U) and triple-stranded poly(A)2poly(U)), DNA G-quadruplex structures (G4s), and bovine serum albumin (BSA) protein. Absorbance and fluorescence titrations, mass spectrometry together with melting and viscometry tests show significant differences in the binding features between silver and gold compounds. [Au(EIA)2]Cl covalently binds BSA. It is here evidenced that the selectivity is high: low affinity and external binding for all polynucleotides and G4s are found. Conversely, in the case of [Ag(EIA)2]Cl, the binding to BSA is weak and relies on electrostatic interactions. [Ag(EIA)2]Cl strongly/selectively interacts only with double strands by a mechanism where intercalation plays the major role, but groove binding is also operative. The absence of an interaction with triplexes indicates the major role played by the geometrical constraints to drive the binding mode.

Protein-metallodrugs interactions: Effects on the overall protein structure and characterization of Au, Ru and Pt binding sites.

The effects of metalation process by inorganic compounds containing Au, Pt and Ru on protein structure and on conformation and flexibility of the residues involved in the metal compound binding have been here investigated by analysing 204 structures of protein/metallodrug adducts and the corresponding metal-free forms. The overall structure of the proteins is not significantly affected by the metal label. 162 non-redundant protein residues involved in Au, Pt and Ru coordination have been identified. In the metal-free protein structures these residues often belong to α-helical regions and show low flexibility. They do not necessarily belong to outer layers of the protein structure. In the majority of the adducts, the side chains of these residues adopt a conformation that is similar to that observed in the metal-free protein. The metal coordination reduces their solvent accessible surface area without altering their overall flexibility. These results could be useful for the prediction of residues able to bind Au, Pt and Ru compounds.

The interactions of the ruthenium(II)-cymene complexes with lysozyme and cytochrome c.

The reactions of four cymene-capped ruthenium(II) compounds with pro-apoptotic protein, cytochrome c (Cyt), and anti-proliferative protein lysozyme (Ly) in carbonate buffer were investigated by ESI-MS, UV-vis absorption, and CD spectroscopy. The complexes with two chloride ligands (C2 and C3) were more reactive toward proteins than those with only one (C1 and C4), and the complex with S,N-chelating ligand (C4) was less reactive than one with O,N-chelating ligand (C1). Dehalogenated complexes are most likely species, initially coordinating proteins for all tested complexes. During the time, protein adducts vividly exchanged non-arene organic ligand L with CO32- and OH-, while cymene moiety was retained. In water, only dehalogenated adducts were identified suggesting that in vivo, in the presence of various anions, dynamic ligand exchange could generate different intermediate protein species. Although all complexes reduced Cyt, the reduction was not dependent on their reactivity to protein, implying that initially noncovalent binding to Cyt occurs, causing its reduction, followed by coordination to protein. Cyt reduction was accompanied with rupture of ferro-Met 80 and occupation of this hem coordination site by a histidine His-33/26. Therefore, in Cyt with C2 and C3, less intensive reduction of hem iron leaves more unoccupied target residues for Ru coordination, leading to more efficient formation of covalent adducts, in comparison to C1 and C4. This study contributes to development of new protein-targeted Ru(II) cymene complexes, and to the design of new cancer therapies based on targeted delivery of Ru(II) arene complexes bound on pro-apoptotic/anti-proliferative proteins as vehicles.

Structural and solution chemistry, antiproliferative effects, and serum albumin binding of three pseudohalide derivatives of auranofin.

Three pseudohalide analogues of the established gold drug auranofin (AF hereafter), of general formula Au(PEt3)X, i.e. Au(PEt3)CN, Au(PEt3)SCN and Au(PEt3)N3 (respectively denoted as AFCN, AFSCN and AFN3), were prepared and characterized. The crystal structure was solved for Au(PEt3)SCN highlighting the classical linear geometry of the 2-coordinate gold(I) center. The solution behaviour of the compounds was then comparatively analysed through 31PNMR providing evidence for an acceptable stability under physiological-like conditions. Afterward, the reaction of these gold compounds with bovine serum albumin (BSA) and consequent adduct formation was investigated by 31PNMR. For all the studied gold compounds, the [Au(PEt3)]+ moiety was identified as the reactive species in metal/protein adducts formation. The cytotoxic effects of the complexes were subsequently measured in comparison to AF against a representative colorectal cancer cell line and found to be still relevant and roughly similar in the three cases though far weaker than those of AF. These results show that the nature of the anionic ligand can modulate importantly the pharmacological action of the gold-triethylphosphine moiety, affecting the cytotoxic potency. These aspects may be further explored to improve the pharmacological profiles of this family of metal complexes.

Preparation, structure, cytotoxicity and mechanism of action of ferritin-Pt(II) terpyridine compound nanocomposites.

AIM: A Pt(II)-terpyridine compound, bearing two piperidine substituents at positions 2 and 2' of the terpyridine ligand (1), is highly cytotoxic and shows a mechanism of action distinct from cisplatin. 1 has been incorporated within the ferritin nanocage (AFt). MATERIALS & METHODS: Spectroscopic and crystallographic data of the Pt(II)-AFt nanocomposite have been collected and in vitro anticancer activity has been explored using cancer cells. RESULTS: Pt(II)-containing fragments bind His49, His114 and His132. Pt(II)-AFt nanocomposite is less cytotoxic than 1, but it is more toxic than cisplatin at high concentrations. The Pt(II)-AFt nanocomposite triggers necrosis in cancer cells, as free 1 does. CONCLUSION: Pt(II)-AFt nanocomposites are promising vehicles to deliver Pt-based drugs to cancer cells.

Caged noble metals: Encapsulation of a cytotoxic platinum(II)-gold(I) compound within the ferritin nanocage.

The encapsulation of Pt and Au-based anticancer agents within a protein cage is a promising way to enhance the selectivity of these potential drugs. Here a cytotoxic organometallic compound containing platinum(II) and gold(I) has been encapsulated within a ferritin nanocage (AFt). Inductively plasma coupled mass spectrometry data, collected to evaluate the amount of Pt and Au within the cage, indicate disruption of the starting heterobimetallic complex upon encapsulation within the nanocage. The drug-loaded protein (Pt(II)/Au(I)-AFt) has been characterized by UV-Vis spectroscopy, circular dichroism and X-ray diffraction analysis. Data indicate that the protein maintains its fold upon encapsulation of the metallodrug and that Au(I) and Pt(II)-containing fragments are encapsulated within the AFt cage, with Au(I) ion that binds the side chain of Cys126 and Pt(II) in the bulk, respectively. The in vitro cytotoxicity of Pt(II)Au(I)-AFt, as well as that of the free heterobimetallic complex, has been comparatively evaluated on human cervix and breast cancer cells and against cardiomyoblasts and keratinocytes non-tumorigenic cells. Our data demonstrate that it is possible to obtain a protein nanocarrier containing both Pt and Au atoms starting from a bimetallic compound, opening the way for the design and development of new potential drugs based on protein nanocarriers.

Protein Metalation by Anticancer Metallodrugs: A Joint ESI MS and XRD Investigative Strategy.

Interactions of metal-based drugs with proteins and consequent adduct formation (the so-called "protein metalation" process) play a key role in the mode of action of several anticancer agents and in determining their toxicological profile. Through a novel investigative strategy grounded on the combined use of electrospray ionization mass spectrometry (ESI MS) and biological macromolecule X-ray crystallography we show that it is possible to clarify in depth the metalation process of small model proteins; a number of instructive examples are provided. Recently, this kind of investigative approach has been extended to bigger proteins such as human serum albumin and horse spleen ferritin, with rather encouraging results. Overall, by application of this strategy, metalation of proteins caused by anticancer metallodrugs can be disclosed in the molecular detail.

Organogold(III) compounds as experimental anticancer agents: chemical and biological profiles.

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.

Interactions of the organogold(III) compound Aubipyc with the copper chaperone Atox1: a joint mass spectrometry and circular dichroism investigation.

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.