Interactions with DNA Models of the Oxaliplatin Analog (cis-1,3-DACH)PtCl2.

It is generally accepted that adjacent guanine residues in DNA are the primary target for platinum antitumor drugs and that differences in the conformations of the Pt-DNA adducts can play a role in their antitumor activity. In this study, we investigated the effect of the carrier ligand cis-1,3-diaminocyclohexane (cis-1,3-DACH) upon formation, stability, and stereochemistry of the (cis-1,3-DACH)PtG2 and (cis-1,3-DACH)Pt(d(GpG)) adducts (G = 9-EthlyGuanine, guanosine, 5'- and 3'-guanosine monophosphate; d(GpG) = deoxyguanosil(3'-5')deoxyguanosine). A peculiar feature of the cis-1,3-DACH carrier ligand is the steric bulk of the diamine, which is asymmetric with respect to the Pt-coordination plane. The (cis-1,3-DACH)Pt(5'GMP)2 and (cis-1,3-DACH)Pt(3'GMP)2 adducts show preference for the ΛHT and ∆HT conformations, respectively (HT stands for Head-to-Tail). Moreover, the increased intensity of the circular dichroism signals in the cis-1,3-DACH derivatives with respect to the analogous cis-(NH3)2 species could be a consequence of the greater bite angle of the cis-1,3-DACH carrier ligand with respect to cis-(NH3)2. Finally, the (cis-1,3-DACH)Pt(d(GpG)) adduct is present in two isomeric forms, each one giving a pair of H8 resonances linked by a NOE cross peak. The two isomers were formed in comparable amounts and had a dominance of the HH conformer but with some contribution of the ΔHT conformer which is related to the HH conformer by having the 3'-G base flipped with respect to the 5'-G residue.

Interference between copper transport systems and platinum drugs.

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 testicular, ovarian and colorectal carcinomas, sarcomas, and lymphomas. Together with other platinum-based drugs, cisplatin 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 systems, including endocytosis and some active or facilitated transport mechanisms, are currently proposed to play a pivotal role in the uptake of platinum-based drugs. In this review, an updated view of the current literature regarding the intracellular transport and processing of cisplatin will be presented, with special emphasis on the plasma membrane copper permease CTR1, the Cu-transporting ATPases, ATP7A and ATP7B, located in the trans-Golgi network, and the soluble copper chaperone ATOX1. Their role in eliciting cisplatin efficacy and their exploitation as pharmacological targets will be addressed.

Improvement of Kiteplatin Efficacy by a Benzoato Pt(IV) Prodrug Suitable for Oral Administration.

Kiteplatin, [PtCl2(cis-1,4-DACH)] (DACH = diaminocyclohexane), contains an isomeric form of the oxaliplatin diamine ligand trans-1R,2R-DACH and has been proposed as a valuable drug candidate against cisplatin- and oxaliplatin-resistant tumors, in particular, colorectal cancer. To further improve the activity of kiteplatin, it has been transformed into a Pt(IV) prodrug by the addition of two benzoato groups in the axial positions. The new compound, cis,trans,cis-[PtCl2(OBz)2(cis-1,4-DACH)] (1; OBz = benzoate), showed cytotoxic activity at nanomolar concentration against a wide panel of human cancer cell lines. Based on these very promising results, the investigation has been extended to the in vivo activity of compound 1 in a Lewis Lung Carcinoma (LLC) model and its suitability for oral administration. Compound 1 resulted to be remarkably stable in acidic conditions (pH 1.5 to mimic the stomach environment) undergoing a drop of the initial concentration to ~60% of the initial one only after 72 h incubation at 37 °C; thus resulting amenable for oral administration. Interestingly, in a murine model (2·106 LLC cells implanted i.m. into the right hind leg of 8-week old male and female C57BL mice), a comparable reduction of tumor mass (~75%) was observed by administering compound 1 by oral gavage and the standard drug cisplatin by intraperitoneal injection, thus indicating that, indeed, there is the possibility of oral administration for this dibenzoato prodrug of kiteplatin. Moreover, since the mechanism of action of Pt(IV) prodrugs involves an initial activation by chemical reduction to cytotoxic Pt(II) species, the reduction of 1 by two bioreductants (ascorbic acid/sodium ascorbate and glutathione) was investigated resulting to be rather slow (not complete after 120 h incubation at 37 °C). Finally, the neurotoxicity of 1 was evaluated using an in vitro assay.

19 F NMR Allows the Investigation of the Fate of Platinum(IV) Prodrugs in Physiological Conditions.

PtIV prodrugs can overcome resistance and side effects of conventional PtII anticancer therapies. By 19 F-labeling of a PtIV prodrug (Pt-FBA, FBA=p-fluorobenzoate), the activation under physiological conditions could be investigated. Unlike single-electron reductants, multi-electron agents can efficiently promote the two electrons reduction of PtIV to PtII . The activation of Pt-FBA in cell lysate is highly dependent upon the type of cancer cells. When administered to E. coli, Pt-FBA is reduced intracellularly and free FBA can shuttle out of the cell. The reduction rate greatly increases by inducing metallothionein overexpression and is lowered by addition of ZnII ions. When injected into mice, Pt-FBA undergoes fast reduction in the bloodstream accompanied by metabolic degradation of FBA; nevertheless, unreduced Pt-FBA can accumulate to detectable levels in liver and kidneys. The 19 F NMR approach has the advantage of avoiding the interference of all background signals.

New Oxaliplatin-Pyrophosphato Analogs with Improved In Vitro Cytotoxicity.

Two new Pt(II)-pyrophosphato complexes containing the carrier ligands cis-1,3-diaminocyclohexane (cis-1,3-DACH) and trans-1,2-diamine-4-cyclohexene (1,2-DACHEX), variants of the 1R,2R-diaminocyclohexane ligand present in the clinically used Pt-drug oxaliplatin, have been synthesized with the aim of developing new potential antitumor drugs with high bone tropism. The complexes are more stable at physiological pH than in acid conditions, with Na2[Pt(pyrophosphato)(cis-1,3-DACH)] (1) slightly more stable than [Pt(dihydrogenpyrophosphato)(1,2-DACHEX)] (2). The greater reactivity at acidic pH ensures a greater efficacy at the tumor site. Preliminary NMR studies indicate that 1 and 2 react slowly with 5'-GMP (used as a model of nucleic acids), releasing the pyrophosphate ligand and affording the bis 5'-GMP adduct. In vitro cytotoxicity assays performed against a panel of four human cancer cell lines have shown that both compounds are more active than oxaliplatin. Flow cytometry studies on HCT116 cells showed that the pyrophosphato compounds with the non-classical 1,3- and 1,4-diaminocyclohexane ligands (1 and 4) are the most capable to induce cells' death by apoptosis and necrosis.

Effect of in vivo post-translational modifications of the HMGB1 protein upon binding to platinated DNA: a molecular simulation study.

Cisplatin is one of the most widely used anticancer drugs. Its efficiency is unfortunately severely hampered by resistance. The High Mobility Group Box (HMGB) proteins may sensitize tumor cells to cisplatin by specifically binding to platinated DNA (PtDNA) lesions. In vivo, the HMGB/PtDNA binding is regulated by multisite post-translational modifications (PTMs). The impact of PTMs on the HMGB/PtDNA complex at atomistic level is here investigated by enhanced sampling molecular simulations. The PTMs turn out to affect the structure of the complex, the mobility of several regions (including the platinated site), and the nature of the protein/PtDNA non-covalent interactions. Overall, the multisite PTMs increase significantly the apparent synchrony of all the contacts between the protein and PtDNA. Consequently, the hydrophobic anchoring of the side chain of F37 between the two cross-linked guanines at the platinated site-a key element of the complexes formation - is more stable than in the complex without PTM. These differences can account for the experimentally measured greater affinity for PtDNA of the protein isoforms with PTMs. The collective behavior of multisite PTMs, as revealed here by the synchrony of contacts, may have a general significance for the modulation of intermolecular recognitions occurring in vivo.

Platinum(IV) Complexes of trans-1,2-diamino-4-cyclohexene: Prodrugs Affording an Oxaliplatin Analogue that Overcomes Cancer Resistance.

Six platinum(IV) compounds derived from an oxaliplatin analogue containing the unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX), in place of the 1,2-diaminocyclohexane, and a range of axial ligands, were synthesized and characterized. The derivatives with at least one axial chlorido ligand demonstrated solvent-assisted photoreduction. The electrochemical redox behavior was investigated by cyclic voltammetry; all compounds showed reduction potentials suitable for activation in vivo. X-ray photoelectron spectroscopy (XPS) data indicated an X-ray-induced surface reduction of the Pt(IV) substrates, which correlates with the reduction potentials measured by cyclic voltammetry. The cytotoxic activity was assessed in vitro on a panel of human cancer cell lines, also including oxaliplatin-resistant cancer cells, and compared with that of the reference compounds cisplatin and oxaliplatin; all IC50 values were remarkably lower than those elicited by cisplatin and somewhat lower than those of oxaliplatin. Compared to the other Pt(IV) compounds of the series, the bis-benzoate derivative was by far (5-8 times) the most cytotoxic showing that low reduction potential and high lipophilicity are essential for good cytotoxicity. Interestingly, all the complexes proved to be more active than cisplatin and oxaliplatin even in three-dimensional spheroids of A431 human cervical cancer cells.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.

Reaction of Histone H1 with trans-Platinum Complexes and the Effect on DNA Platination.

Transplatin is an inactive platinum drug; however, a number of analogues, such as trans-EE and trans-PtTz, demonstrate promising antitumor activity in vitro and in vivo. Although the ultimate target is nuclear DNA, increasing evidence indicate that proteins also play important roles in the display of antitumor activity. The linker histone H1 is situated by the portal between the unwrapped DNA and the nucleosome core. Our recent study revealed that H1 can readily react with cisplatin, and the adducts tend to form ternary complexes with DNA. In this work, we have investigated the reaction of histone H1 with two antitumor-active trans-oriented complexes, trans-EE and trans-PtTz, and the effect of H1 upon the platination of DNA. The results show that trans-platinum drugs are much more reactive than cisplatin toward H1. Interestingly, in addition to the expected bidentate adducts (by displacement of the two labile chlorido ligands), also a tridentate adduct can be formed by displacement of one nonlabile carrier ligand of trans-EE or trans-PtTz. The trans-Pt/H1 adducts can then react with DNA and generate protein-Pt-DNA ternary complexes. Additionally, platinum can be transferred from trans-Pt/H1 adducts to DNA, generating binary trans-Pt/DNA complexes. Such a transfer of the platinum agent to DNA was not observed in the reaction of cisplatin. Furthermore, the detailed investigation carried out on a model peptide indicates that H1 promotes the DNA platination by trans- EE, while it reduces that of trans-PtTz and cisplatin. These results suggest that H1 can play a key role in the DNA platination and modulate the efficacy of different platinum agents.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Aggregation Pathways of Native-Like Ubiquitin Promoted by Single-Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium.

Ubiquitin-positive protein aggregates are biomarkers of neurodegeneration, but the molecular mechanism responsible for their formation and accumulation is still unclear. Possible aggregation pathways of human ubiquitin (hUb) promoted by both intrinsic and extrinsic factors, are here investigated. By a computational analysis, two different hUb dimers are indicated as possible precursors of amyloid-like structures, but their formation is disfavored by an electrostatic repulsion involving Glu16 and other carboxylate residues present at the dimer interface. Experimental data on the E16V mutant of hUb shows that this single-point mutation, although not affecting the overall protein conformation, promotes protein aggregation. It is sufficient to shift the same mutation by only two residues (E18V) to regain the behavior of wild-type hUb. The neutralization of Glu16 negative charge by a metal ion and a decrease of the dielectric constant of the medium by addition of trifluoroethanol (TFE), also promote hUb aggregation. The outcomes of this research have important implications for the prediction of physiological parameters that favor aggregate formation.

Differential Reactivity of Metal Binding Domains of Copper ATPases towards Cisplatin and Colocalization of Copper and Platinum.

The Menkes (MNK) and Wilson (WLN) disease proteins are two P-type ATPases responsible for active Cu efflux. These ATPases are also associated with resistance to cisplatin. In this work, different metal-binding domains (MBDs) of ATPases (9 out of 12 domains) were compared based on their reactivity towards cisplatin. The reaction rates of the MBDs can be largely different; the reaction of MNK6 is about six times faster than that of WLN2. Copper coordination favors the platination of the MBDs to different extents. The rate of platination was generally greater for holo-MBDs than for apo-MBDS (particularly in the case of WLN4 and WLN2); however, it was negligibly affected in the case of MNK6. Interestingly, the platinum binding weakens the CuI coordination, but does not expel the copper ion from MBDs. The latter results nicely explain the inhibitory effect of Cu upon the cisplatin translocation promoted by Cu-ATPases and can help in understanding how copper levels can modulate the sensitivity of cancer cells to platinum chemotherapy.

Multi-Acting Mitochondria-Targeted Platinum(IV) Prodrugs of Kiteplatin with α-Lipoic Acid in the Axial Positions.

Platinum(II) drugs are activated intracellularly by aquation of the leaving groups and then bind to DNA, forming DNA adducts capable to activate various signal-transduction pathways. Mostly explored in recent years are Pt(IV) complexes which allow the presence of two additional ligands in the axial positions suitable for the attachment of other cancer-targeting ligands. Here we have extended this strategy by coordinating in the axial positions of kiteplatin ([PtCl2(cis-1,4-DACH)], DACH = Diaminocyclohexane) and its CBDCA (1,1-cyclobutanedicarboxylate) analogue the antioxidant α-Lipoic acid (ALA), an inhibitor of the mitochondrial pyruvate dehydrogenase kinase (PDK). The new compounds (cis,trans,cis-[Pt(CBDCA)(ALA)2(cis-1,4-DACH)], 2, and cis,trans,cis-[PtCl2(ALA)2(cis-1,4-DACH)], 3), after intracellular reduction, release the precursor Pt(II) species and two molecules of ALA. The Pt residue is able to target DNA, while ALA could act on mitochondria as activator of the pyruvate dehydrogenase complex, thus suppressing anaerobic glycolysis. Compounds 2 and 3 were tested in vitro on a panel of five human cancer cell lines and compared to cisplatin, oxaliplatin, and kiteplatin. They proved to be much more effective than the reference compounds, with complex 3 most effective in 3D spheroid tumor cultures. Notably, treatment of human A431 carcinoma cells with 2 and 3 did not determine increase of cellular ROS (usually correlated to inhibition of mitochondrial PDK) and did not induce a significant depolarization of the mitochondrial membrane or alteration of other morphological mitochondrial parameters.

Novel Kiteplatin Pyrophosphate Derivatives with Improved Efficacy.

Two new Pt(II) derivatives of kiteplatin ([PtCl2(cis-1,4-DACH)]) with pyrophosphate as carrier ligand, one mononuclear (1) and one dinuclear (2), were synthesized with the aim of potentiating the efficacy of kiteplatin. Complex 1 resulted to be remarkably stable at physiological pH, but it undergoes a fast hydrolysis reaction at acidic pH releasing free pyrophosphate and (aquated) kiteplatin. The dinuclear compound 2 resulted to be less stable than 1 at both neutral and acidic pH forming 1 and (aquated) kiteplatin as first step. Both compounds (1 and 2) do not react as such with 5'-GMP, whereas their hydrolysis products readily form adducts with the nucleotide. The in vitro cytotoxicity assays against a panel of six human cancer cell lines showed that complex 2 affects cancer cell viability even at nanomolar concentrations. The cytotoxic activity of 2 is greater (up to 2 orders of magnitude) than that of cisplatin, oxaliplatin, and kiteplatin, whereas the mononuclear complex 1 has shown a cytotoxic activity comparable to that of oxaliplatin and kiteplatin, but higher than cisplatin. The latter result is not surprising, since the presence of two negative charges reduces the uptake of 1 into the tumor cells as compared to the neutral compound 2. The remarkable activity of 2 against the pancreatic cell line BxPC3 (average IC50 = 0.07 µM) deserves further investigation.

Novel Antitumor Platinum(II) Conjugates Containing the Nonsteroidal Anti-inflammatory Agent Diclofenac: Synthesis and Dual Mechanisms of Antiproliferative Effects.

One concept how to improve anticancer effects of conventional metallodrugs consists in conjugation of these compounds with other biologically (antitumor) active agents, acting by a different mechanism. Here, we present synthesis, biological effects, and mechanisms of action of new Pt(II) derivatives containing one or two nonsteroidal anti-inflammatory diclofenac (DCF) ligands also known for their antitumor effects. The antiproliferative properties of these metallic conjugates show that these compounds are potent and cancer cell selective cytotoxic agents exhibiting activity in cisplatin resistant and the COX-2 positive tumor cell lines. One of these compounds, compound 3, in which DCF molecules are coordinated to Pt(II) through their carboxylic group, is more potent than parental conventional Pt(II) drug cisplatin, free DCF and the congeners of 3 in which DCF ligands are conjugated to Pt(II) via a diamine. The potency of 3 is due to several factors including enhanced internalization that correlates with enhanced DNA binding and cytotoxicity. Mechanistic studies show that 3 combines multiple effects. After its accumulation in cells, it releases Pt(II) drug capable of binding/damaging DNA and DCF ligands, which affect distribution of cells in individual phases of the cell cycle, inhibit glycolysis and lactate transport, collapse mitochondrial membrane potential, and suppress the cellular properties characteristic of metastatic progression.

An Updated View of Translocator Protein (TSPO).

Decades of study on the role of mitochondria in living cells have evidenced the importance of the 18 kDa mitochondrial translocator protein (TSPO), first discovered in the 1977 as an alternative binding site for the benzodiazepine diazepam in the kidneys. This protein participates in a variety of cellular functions, including cholesterol transport, steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and cell proliferation. Thus, TSPO has become an extremely attractive subcellular target for the early detection of disease states that involve the overexpression of this protein and the selective mitochondrial drug delivery. This special issue was programmed with the aim of summarizing the latest findings about the role of TSPO in eukaryotic cells and as a potential subcellular target of diagnostics or therapeutics. A total of 9 papers have been accepted for publication in this issue, in particular, 2 reviews and 7 primary data manuscripts, overall describing the main advances in this field.

Activation of Platinum(IV) Prodrugs by Cytochrome c and Characterization of the Protein Binding Sites.

Platinum(IV) complexes generally require reduction to reactive Pt(II) species to exert their chemotherapeutic activity. The process of reductive activation of (15)N-labeled (OC-6-43)-bis(acetato)diamminedichloridoplatinum(IV), in the presence of nicotinamide adenine dinucleotide (NADH) and horse heart cytochrome c (cyt c), was monitored by (1)H,(15)N-HSQC NMR spectroscopy and protein digestion experiments. It has been shown that cyt c plays a catalytic role in the transfer of two reducing equivalents from NADH to Pt(IV) species. Noncovalent interactions between reduced monoaqua cisplatin (cis-[PtCl((15)NH3)2(H2O)](+)) and the protein, in the proximity of the heme cofactor, and also covalent binding of platinum to the protein region around Met65 and Met80 take place.

Photoactivation of Diiodido-Pt(IV) Complexes Coupled to Upconverting Nanoparticles.

The preparation, characterization, and surface modification of upconverting lanthanide-doped hexagonal NaGdF4 nanocrystals attached to light sensitive diiodido-Pt(IV) complexes is presented. The evaluation for photoactivation and cytotoxicity of the novel carboxylated diiodido-Pt(IV) cytotoxic prodrugs by near-infrared (NIR) light (λ = 980 nm) is also reported. We attempted two different strategies for attachment of light-sensitive diiodido-Pt(IV) complexes to Yb,Er- and Yb,Tm-doped ß-NaGdF4 upconverting nanoparticles (UCNPs) in order to provide nanohybrids, which offer unique opportunities for selective drug activation within the tumor cells and subsequent spatiotemporal controlled drug release by NIR-to-visible light-upconversion: (A) covalent attachment of the Pt(IV) complex via amide bond formation and (B) carboxylate exchange of oleate on the surface of the UCNPs with diiodido-Pt(IV) carboxylato complexes. Initial feasibility studies showed that NIR applied by a 980 nm laser had only a slight effect on the stability of the various diiodido-Pt(IV) complexes, but when UCNPs were present more rapid loss of the ligand-metal-charge transfer (LMCT) bands of the diiodido-Pt(IV) complexes was observed. Furthermore, Pt released from the Pt(IV) complexes platinated calf-thymus DNA (ct-DNA) more rapidly when NIR was applied compared to dark controls. Of the two attachment strategies, method A with the covalently attached diiodido-Pt(IV) carboxylates via amide bond formation proved to be the most effective method for generating UCNPs that release Pt when irradiated with NIR; the released Pt was also able to bind irreversibly to calf thymus DNA. Nonetheless, only ca. 20% of the Pt on the surface of the UCNPs was in the Pt(IV) oxidation state, the rest was Pt(II), indicating chemical reduction of the diiodido-Pt(IV) prodrug by the UCNPs. Cytotoxicity studies with the various UCNP-Pt conjugates and constructs, tested on human leukemia HL60 cells in culture, indicated a substantial increase in cytotoxicity when modified UCNPs were combined with five rounds of 30 min irradiation with NIR compared to dark controls, but NIR alone also had a significant cytotoxic effect at this duration.

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.

Synthesis, Characterization, and Cytotoxicity of the First Oxaliplatin Pt(IV) Derivative Having a TSPO Ligand in the Axial Position.

The first Pt(IV) derivative of oxaliplatin carrying a ligand for TSPO (the 18-kDa mitochondrial translocator protein) has been developed. The expression of the translocator protein in the brain and liver of healthy humans is usually low, oppositely to steroid-synthesizing and rapidly proliferating tissues, where TSPO is much more abundant. The novel Pt(IV) complex, cis,trans,cis-[Pt(ethanedioato)Cl{2-(2-(4-(6,8-dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-a]pyridin-2-yl)phenoxy)acetate)-ethanolato}(1R,2R-DACH)] (DACH = diaminocyclohexane), has been fully characterized by spectroscopic and spectrometric techniques and tested in vitro against human MCF7 breast carcinoma, U87 glioblastoma, and LoVo colon adenocarcinoma cell lines. In addition, affinity for TSPO (IC50 = 18.64 nM), cellular uptake (ca. 2 times greater than that of oxaliplatin in LoVo cancer cells, after 24 h treatment), and perturbation of cell cycle progression were investigated. Although the new compound was less active than oxaliplatin and did not exploit a synergistic proapoptotic effect due to the presence of the TSPO ligand, it appears to be promising in a receptor-mediated drug targeting context towards TSPO-overexpressing tumors, in particular colorectal cancer (IC50 = 2.31 µM after 72 h treatment).