Cisplatin, or cis-diamminedichloridoplatinum(II) cis-[PtCl2(NH3)2], is a platinum-based anticancer drug largely used for the treatment of various types of cancers, including ovarian and colorectal carcinomas, sarcomas, and lymphomas. Together with other platinum-based drugs, it triggers malignant cell death by binding to nuclear DNA, which appears to be the ultimate target. In addition to passive diffusion across the cell membrane, other transport mechanisms, including endocytosis and some active or facilitated transport, are currently proposed to play a pivotal role in the uptake of platinum-based drugs. In this microreview, we will give an updated view of the current literature regarding cisplatin transport and processing inside the cell, with special emphasis on the membrane copper transporter Ctr1 and the soluble copper chaperone Atox1.

Integrating carbon nanoparticles (CNPs) with proteins to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science. The comprehension of CNP-protein interactions requires the still-missing identification and characterization of the 'binding pocket' for the CNPs. Here, using Lysozyme and C60 as model systems and NMR chemical shift perturbation analysis, a protein-CNP binding pocket is identified unambiguously in solution and the effect of the binding, at the level of the single amino acid, is characterized by a variety of experimental and computational approaches. Lysozyme forms a stoichiometric 1:1 adduct with C60 that it is dispersed monomolecularly in water. Lysozyme maintains its tridimensional structure upon interaction with C60 and only a few identified residues are perturbed. The C60 recognition is highly specific and localized in a well-defined pocket.

It has been proposed that the well-studied monofunctional platinum complex cis-[PtCl(NH3)2(py)]+ (cDPCP) forms DNA adducts similar to those of the trans platinum complex trans-[PtCl2(NH3)(py)] (ampyplatin, py=pyridine). Thus this latter could be the active form of cDPCP. Detailed studies on the mechanism of ampyplatin action were performed in this work. Results indicate that ampyplatin has significantly higher antiproliferative activity than cDPCP and is comparable to cisplatin. Cellular uptake experiments indicate that ampyplatin can be efficiently accumulated in A549 cancer cells. Binding of ampyplatin to DNA mainly produces monofunctional adducts; remarkably, these adducts can be recognized by the HMGB1 protein. Kinetic studies on the reaction with GMP indicate that the reactivity of ampyplatin is much lower than that of transplatin and is more similar to that of trans-[PtCl2{E-HN=C(Me)OMe}2] (trans-EE), a widely investigated antitumor active trans-oriented platinum complex. In addition, the hydrolysis of ampyplatin is significantly suppressed, whereas the hydrolysis of the mono-GMP adduct is highly enhanced. These results indicate that the mechanism of ampyplatin differs not only from that of antitumor inactive transplatin but also from that of antitumor active trans-EE and this could account for the remarkable activity of parent cDPCP.

Copper (Cu) transporters emerged as key factors at the basis of the biological response to antitumor platinum (Pt) drugs, which are among the most potent and broadly used chemotherapeutics. ATP7A and ATP7B (the Menkes and Wilson disease proteins, respectively) appear to be implicated in promoting tumor cell resistance to cisplatin. Cu-ATPases could bind the drug and, with the alleged involvement of the chaperone ATOX1, contribute to cell detoxification and survival. Here, we report the spectroscopic characterization of cisplatin binding to ATOX1 and MNK1, the first metal-binding domain of ATP7A, in the presence of the physiological reducing agent glutathione, a sulfur-containing molecule responsible for the majority of Pt detoxification in the cytosol. Under conditions mimicking the cellular environment, we show that cisplatin transfer from ATOX1 to MNK1 does not occur at a detectable rate. These results appear to contradict other literature data which, however, were obtained in the presence of exogenous reducing agents such as tris(2-carboxyethyl)phosphine (TCEP) having good coordinating ability for soft metal ions (such as Pt) and strong trans-labilizing effect. A better understanding of Pt drug processing by Cu trafficking proteins under physiological conditions may help to answer key issues, such as drug availability in tumor cells and resistance.

Zinc ions bridging two ubiquitin molecules (with His68 at the interface) contribute to select a subset of conformers from the noncovalent dimer ensemble, thus restricting quaternary structure dynamics, which hampers apo-protein crystallization. The type of selected conformer is shown to determine the crystal packing which varies from orthorhombic to cubic symmetry.

Biological targets, such as proteins and nucleic acids, are chiral, therefore stereoisomers of chiral molecules interact with these targets differently and, indeed, the antitumor drug oxaliplatin contains only one enantiomer (R,R) of its 1,2-cyclohexanediamine (DACH) ligand. In this review article we illustrate the effect of chirality in platinum drugs in relation to different aspects spanning from cytotoxicity to mutagenicity, from differences in the reaction with DNA and processing of DNA lesions to gene expression and proteomic profile, to conclude with a section on the use of platinum compounds with chiral amines to investigate non-covalent interactions in adducts of platinum drugs with nucleotides and DNA. Unlike the deep understanding of the interactions at a molecular level which has allowed us to interpret the different antitumor activity and mutagenicity of DACH enantiomers and to propose an explanation for the particularly high efficacy of cisplatin toward the testis tumor, it is noted that “omics” investigations are still scanty and a reassessment of chirality effects, through molecular profiling technologies, would be timely as well as appropriate.

Cadmium (Cd) is an environmental contaminant, highly toxic to humans. This biologically non-essential element accumulates in the body, especially in the kidney, liver, lung and brain and can induce several toxic effects, depending on the concentration and the exposure time. Cd has been linked to Alzheimer's disease (AD) as a probable risk factor, as it shows higher concentrations in brain tissues of AD patients than in healthy people, its implication in the formation of neurofibrillary tangles and in the aggregation process of amyloid beta peptides (AβPs). AβPs seem to have toxic properties, particularly in their aggregated state; insoluble AβP forms, such as small and large aggregates, protofibrils and fibrils, appear to be implicated in the pathogenesis of AD. In our study, we have evaluated the effect of Cd, at different concentrations, both on the AβP1-42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30 % cholesterol and on the secondary structure of AβP1-42 in aqueous environment. Cadmium is able to interact with the AβP1-42 peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both decreasing AβP1-42 channel frequency and in solution forming large and amorphous aggregates prone to precipitate. These experimental observations suggesting a toxic role for Cd strengthen the hypothesis that Cd may interact directly with AβPs and may be a risk factor in AD.

The aim of this study was to evaluate chitosan (CS)-, glycol chitosan (GCS)- and corresponding thiomer-based nanoparticles (NPs) for delivering dopamine (DA) to the brain by nasal route. Thus, the polyanions tripolyphosphate and sulfobutylether-β-cyclodextrin (SBE-β-CD), respectively, were used as polycation crosslinking agents and SBE-β-CD also in order to enhance the DA stability. The most interesting formulation, containing GCS and SBE-β-CD, was denoted as DA GCS/DA-CD NPs. NMR spectroscopy demonstrated an inclusion complex formation between SBE-β-CD and DA. X-ray photoelectron spectroscopy analysis revealed the presence of DA on the external surface of NPs. DA GCS/DA-CD NPs showed cytotoxic effect toward Olfactory Ensheathing Cells only at higher dosage. Acute administration of DA GCS/DA-CD NPs into the right nostril of rats did not modify the levels of the neurotransmitter in both right and left striatum. Conversely, repeated intranasal administration of DA GCS/DA-CD NPs into the right nostril significantly increased DA in the ipsilateral striatum. Fluorescent microscopy of olfactory bulb after acute administration of DA fluorescent-labeled GCS/DA-CD NPs into the right nostril showed the presence of NPs only in the right olfactory bulb and no morphological tissue damage occurred. Thus, these GCS based NPs could be potentially used as carriers for nose-to-brain DA delivery for the Parkinson's disease treatment.

In Alzheimer's disease (AD), native Aβ protein monomers aggregate through the formation of a variety of water-soluble, toxic oligomers, ultimately leading to insoluble fibrillar deposits. The inhibition of oligomers formation and/or their dissociation into non-toxic monomers, are considered an attractive strategy for the prevention and treatment of AD. A number of studies have demonstrated that small molecules, containing single or multiple (hetero)aromatic rings, can inhibit protein aggregation, being potentially effective in AD treatment. Starting from previously reported data on the antiamyloidogenic activity of a series of 3-hydrazonoindolinones, compound PT2 was selected to deeply investigate the inhibitory mechanism in the Aβ aggregation cascade. We compared data from DLS, 1H, 13C, 15N HSQC NMR, CD, TEM and ThT fluorescence measures to ascertain the interactions with amyloidogenic species formed in vitro during the aggregation process, and confirmed this feature with cell viability tests on HeLa cultured cells. PT2 was effective in disrupting toxic oligomers and mature amyloid fibrils, stabilizing Aβ as non-toxic, β-sheet arranged, ThT-insensitive protofilaments. It also strongly reduced cellular toxicity caused by Aβ and showed good antioxidant properties in two radical scavenging tests. Taken together, these data confirmed that PT2 is a small molecule inhibitor of Aβ oligomerization and toxicity, displaying also additional activity as antioxidant.

Resistance, either at the onset of the treatment or developed after an initial positive response, is a major limitation of antitumor therapy. In the case of platinum-based drugs, copper transporters have been found to interfere with drug trafficking by facilitating the import or favoring the platinum export and inactivation.

The clinical efficacy of the widely used anticancer drug cisplatin is severely limited by the emergence of resistance. This is related to the drug binding to proteins such as the copper influx transporter Ctr1, the copper chaperone Atox1, and the copper pumps ATP7A and ATP7B. While the binding modes of cisplatin to the first two proteins are known, the structural determinants of platinated ATP7A/ATP7B are lacking. Here we investigate the interaction of cisplatin with the first soluble domain of ATP7A. First, we establish by ESI-MS and (1)H, (13)C, and (15)N NMR that, in solution, the adduct is a monomer in which the sulfur atoms of residues Cys19 and Cys22 are cis-coordinated to the [Pt(NH3)2](2+) moiety. Then, we carry out hybrid Car-Parrinello QM/MM simulations and computational spectroscopy calculations on a model adduct based on the NMR structure of the apo protein and featuring the experimentally determined binding mode of the metal ion. These calculations show quantitative agreement with CD spectra and (1)H, (13)C, and (15)N NMR chemical shifts, thus providing a quantitative molecular view of the 3D binding mode of cisplatin to ATP7A. Importantly, the same comparison rules out a variety of alternative models with different coordination modes, that we explored to test the robustness of the computational approach. Using this combined in silico-in vitro approach we provide here for the first time a quantitative 3D atomic view of the platinum binding to the first soluble domain of ATP7A.

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH3)2}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation.

Cisplatin is one of the most used anticancer drugs. Its cellular influx and delivery to target DNA may involve the copper chaperone Atox1 protein. Although the mode of binding is established by NMR spectroscopy measurements in solution-the Pt atom binds to Cys12 and Cys15 while retaining the two ammine groups-the structural determinants of the adduct are not known. Here a structural model by hybrid Car-Parrinello density functional theory-based QM/MM simulations is provided. The platinated site minimally modifies the fold of the protein. The calculated NMR and CD spectral properties are fully consistent with the experimental data. Our in silico/in vitro approach provides, together with previous studies, an unprecedented view into the structural biology of cisplatin-protein adducts.

The cellular uptake of cisplatin and of other platinum-based drugs is mediated by the high-affinity copper transporter Ctr1. The eight-residue long peptide called Mets7 (MTGMKGMS) mimics one of extracellular methionine (Met)-rich motifs of Ctr1. It is an excellent model for investigating the interaction of platinum drugs with Ctr1 under in vitro and in vivo conditions. Some of us have shown that (i) Cisplatin loses all of its ligands upon reaction with Mets7 and the metal ion binds to the three Met residues and completes its coordination shell with a fourth ligand that can be a chloride or a water/hydroxyl oxygen. (ii) Transplatin loses only the chlorido ligands, which are replaced by Met residues. Here, we provide information on the structural determinants of cisplatin/Mets7 and transplatin/Mets7 adducts by computational methods. The predictions are validated against EXAFS, NMR, and CD spectra. While EXAFS gives information restricted to the metal coordination shell, NMR provides information extended to residue atoms around the coordination shell, and finally, CD provides information about the overall conformation of the peptide. This allows us to elucidate the different reaction modes of cisplatin and transplatin toward the peptide, as well as to propose the platinated peptides [PtX]+–(M*TGM*KGM*S) (X = Cl–, OH–) and trans[Pt(NH3)2]2+–(M*TGM*KGMS) as the most relevant species occurring in water solution.

An X-ray investigation has been performed with the aim of characterizing the binding sites of a platinum-based inhibitor (K[PtCl3(DMSO)]) of matrix metalloproteinase-3 (stromelysin-1). The platinum complex targets His224 in the S1' specificity loop, representing the first step in the selective inhibition process.

The human copper protein (hCTR1) is believed to facilitate the cellular uptake of cisplatin. Cisplatin likely binds to the methionine (Met)-rich motifs located in the N-terminus of hCTR1, and ligand exchange would be essential if cisplatin has to pass through the hCTR1 channel. In this work, we investigated the reaction between platinated adducts of a methionine-rich motif of yeast CTR1 (Mets7) and N-acetyl-cysteine (AcCys) or N-acetyl-histidine (AcHis), mimicking metal-binding residues downstream the CTR1 channel. Platination involved two cis-compounds, cisplatin and oxaliplatin, and one monofunctional complex, cis-diammine(pyridine)chloridoplatinum(II) (cDPCP). The reactions were monitored by HPLC and the products were characterized by ESI-MS. The results indicate different reactivities depending upon the platinum complex. The cisplatin/Mets7 adduct reacts readily with both cysteine and histidine (t1/2<2min). In contrast, the oxaliplatin/Mets7 adduct reacts with cysteine but not with histidine, whereas cDPCP/Mets7 adduct reacts with histidine but not with cysteine. Hence, Mets7 adducts of these platinum complexes exhibit different reactivities towards downstream coordinating amino acids. These results suggest that each platinum complex possesses different reactivities and consequently may lead to differences in their cellular distribution and bioactivity.

Cisplatin, carboplatin, and oxaliplatin are widely used anticancer drugs. Their efficacy is strongly reduced by development of cell resistance. Down-regulation of CTR1 and up-regulation of the Cu-ATPases, ATP7A and ATP7B, have been associated to augmented drug resistance. To gain information on translocation of Pt drugs by human Cu-ATPases, we performed electrical measurements on the COS-1 cell microsomal fraction, enriched with recombinant ATP7A, ATP7B, and selected mutants, and adsorbed on a solid supported membrane. The experimental results indicate that Pt drugs activate Cu-ATPases and undergo ATP-dependent translocation in a fashion similar to that of Cu. We then used NMR spectroscopy and ESI-MS to determine the binding mode of these drugs to the first N-terminal metal-binding domain of ATP7A (Mnk1).