Interaction of Copper Trafficking Proteins with the Platinum Anticancer Drug Kiteplatin.

The interaction of metallodrugs with proteins influences their mechanism of action and side effects. In the case of platinum drugs, copper transporters modulate sensitivity and resistance to these anticancer agents. To deepen the knowledge of the structural properties underlying the reactivity of platinum drugs with copper transporters, we studied the interaction of kiteplatin and two of its derivatives with the methionine-rich motif of copper importer Ctr1 and with the dithiol motif of the first domain of Menkes ATPase. Furthermore, cellular uptake and cytotoxicity of the three complexes were evaluated in cisplatin-sensitive and -resistant ovarian cancer cells, comparing the data with those of clinically relevant drugs. Reactivity depends on the tightness of the chelate ring formed by the carrier ligands and the nature of the leaving and entering groups. The results highlight the importance of subtle changes in the platinum coordination sphere that affect drug absorption and intracellular fate.

Duplications of an iron-sulphur tripeptide leads to the formation of a protoferredoxin.

Based on UV-Vis, NMR, and EPR spectroscopies and DFT and molecular dynamics calculations, a model prebiotic [2Fe-2S] tripeptide was shown to accept and donate electrons. Duplications of the tripeptide sequence led to a protoferredoxin with increased stability. Duplications of primitive peptides may have contributed to the formation of contemporary ferredoxins.

Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the reinterpretation of the original crystallographic data put forward by Shabalin et al. (1 Cu occupancy), and could apply to other cases.

Cellular trafficking, accumulation and DNA platination of a series of cisplatin-based dicarboxylato Pt(IV) prodrugs.

A series of Pt(IV) anticancer prodrug candidates, having the equatorial arrangement of cisplatin and bearing two aliphatic carboxylato axial ligands, has been investigated to prove the relationship between lipophilicity, cellular accumulation, DNA platination and antiproliferative activity on the cisplatin-sensitive A2780 ovarian cancer cell line. Unlike cisplatin, no facilitated influx/efflux mechanism appears to operate in the case of the Pt(IV) complexes under investigation, thus indicating that they enter by passive diffusion. While Pt(IV) complexes having lipophilicity comparable to that of cisplatin (negative values of log Po/w) exhibit a cellular accumulation similar to that of cisplatin, the most lipophilic complexes of the series show much higher cellular accumulation (stemming from enhanced passive diffusion), accompanied by greater DNA platination and cell growth inhibition. Even if the Pt(IV) complexes are removed from the culture medium in the recovery process, the level of DNA platination remains very high and persistent in time, indicating efficient storing of the complexes and poor detoxification efficiency.

Structural biology of cisplatin complexes with cellular targets: the adduct with human copper chaperone atox1 in aqueous solution.

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.

Platination of the copper transporter ATP7A involved in anticancer drug resistance.

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.

Cisplatin handover between copper transporters: the effect of reducing agents.

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.

Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B.

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).

AßP1-42 incorporation and channel formation in planar lipid membranes: the role of cholesterol and its oxidation products.

Amyloid beta peptide (AßP) is a natural peptide, normally released into the cerebrospinal fluid (CSF), that plays a key role in Alzheimer's disease. The conversion of the peptide from a native soluble form to a non-native and often insoluble form, such as small and large aggregates, protofibrils and fibrils of AßP appears to be implicated in the pathogenesis of AD. Although the molecular mechanisms of AßP neurotoxicity are not fully understood, a large body of data suggests that the primary target of amyloid peptides is the cell membrane of neurons, that may modulate the structural and functional conversion of AßP into assemblies involved in pathological processes. In our study, we provide a systematic investigation of AßP1-42's ability to incorporate and form channel-like events in membranes of different lipid composition and focus on cholesterol and its oxidation products. We propose that cholesterol and its oxidation products can be considered neuroprotective factors because a) by favouring AßP1-42 insertion into membranes, the fibrillation/clearance balance shifts toward clearance; b) by shifting channel selectivity toward anions, the membrane potential is moved far from the threshold of membrane excitability, thus decreasing the influx of calcium into the cell.