Highly photoactive Ir(III)-Pt(IV) heterometallic conjugates for anticancer therapy.

For the first time, this study reported the photoactivatable activity of Ir(III)-Pt(IV) heterometallic conjugates, which were stable in the dark and activated to release oxaliplatin and Ir within 3 min of irradiation. The conjugates induced apoptosis and immunologic cell death through Pt-DNA binding and reactive oxygen species generation upon irradiation. This work developed photoactivatable heterometallic agents for anticancer therapy.

Compatibility of Nucleobases Containing Pt(II) Complexes with Red Blood Cells for Possible Drug Delivery Applications.

The therapeutic advantages of some platinum complexes as major anticancer chemotherapeutic agents and of nucleoside analogue-based compounds as essential antiviral/antitumor drugs are widely recognized. Red blood cells (RBCs) offer a potential new strategy for the targeted release of therapeutic agents due to their biocompatibility, which can protect loaded drugs from inactivation in the blood, thus improving biodistribution. In this study, we evaluated the feasibility of loading model nucleobase-containing Pt(II) complexes into human RBCs that were highly stabilized by four N-donors and susceptible to further modification for possible antitumor/antiviral applications. Specifically, platinum-based nucleoside derivatives [PtII(dien)(N7-Guo)]2+, [PtII(dien)(N7-dGuo)]2+, and [PtII(dien)(N7-dGTP)] (dien = diethylenetriamine; Guo = guanosine; dGuo = 2'-deoxy-guanosine; dGTP = 5'-(2'-deoxy)-guanosine-triphosphate) were investigated. These Pt(II) complexes were demonstrated to be stable species suitable for incorporation into RBCs. This result opens avenues for the possible incorporation of other metalated nucleobases analogues, with potential antitumor and/or antiviral activity, into RBCs.

The mechanism of antiproliferative activity of the oxaliplatin pyrophosphate derivative involves its binding to nuclear DNA in cancer cells.

(1R,2R-diaminocyclohexane)(dihydropyrophosphato) platinum(II), also abbreviated as RRD2, belongs to a class of potent antitumor platinum cytostatics called phosphaplatins. Curiously, several published studies have suggested significant mechanistic differences between phosphaplatins and conventional platinum antitumor drugs. Controversial findings have been published regarding the role of RRD2 binding to DNA in the mechanism of its antiproliferative activity in cancer cells. This prompted us to perform detailed studies to confirm or rule out the role of RRD2 binding to DNA in its antiproliferative effect in cancer cells. Here, we show that RRD2 exhibits excellent antiproliferative activity in various cancer cell lines, with IC50 values in the low micromolar or submicromolar range. Moreover, the results of this study demonstrate that DNA lesions caused by RRD2 contribute to killing cancer cells treated with this phosphaplatin derivative. Additionally, our data indicate that RRD2 accumulates in cancer cells but to a lesser extent than cisplatin. On the other hand, the efficiency of cisplatin and RRD2, after they accumulate in cancer cells, in binding to nuclear DNA is similar. Our results also show that RRD2 in the medium, in which the cells were cultured before RRD2 accumulated inside the cells, remained intact. This result is consistent with the view that RRD2 is activated by releasing free pyrophosphate only in the environment of cancer cells, thereby allowing RRD2 to bind to nuclear DNA.

Identification and Characterization of DNA-Oxaliplatin Adducts through α-hemolysin Nanopores.

The antitumor effect of Pt-based drugs is determined by their binding activity with deoxyribonucleic acid (DNA), and understanding the reaction process in a systematic manner is crucial. However, existing assays used for DNA-Pt research suffer from several issues, such as complicated sample preparation, preamplification, and expensive instruments, which dramatically limit their practical application. In this study, a novel method was presented to investigate the adducts of DNA and oxaliplatin using an α-hemolysin nanopore sensor. This approach allows for real-time monitoring of the DNA-oxaliplatin condensation process through the detection of nanopore events associated with DNA-oxaliplatin adducts. Specifically, type I and II signals exhibiting specific current characteristics were observed during the process. Typical signals with high frequency were obtained by recording the designed DNA sequence. Furthermore, the production of these signals was confirmed to be independent of homologous adducts. This finding suggests that the DNA-oxaliplatin adduct can serve as a potential sensor for detecting oxaliplatin lesions and multiple types of molecules.

Investigations of cellular copper metabolism in ovarian cancer cells using a ratiometric fluorescent copper dye.

Imbalances in metal homeostasis have been implicated in the progression and drug response of cancer cells. Understanding these changes will enable identification of new treatment regimes and precision medicine approaches to cancer treatment. In particular, there has been considerable interest in the interplay between copper homeostasis and response to platinum-based chemotherapeutic agents. Here, we have studied differences in the Cu uptake and distributions in the ovarian cancer cell line, A2780, and its cisplatin resistant form, A2780.CisR, by measuring total Cu content and the bioavailable Cu pool. Atomic absorption spectroscopy (AAS) revealed a lower total Cu uptake in A2780.CisR compared to A2780 cells. Conversely, live-cell confocal microscopy studies with the ratiometric Cu(I)-sensitive fluorescent dye, InCCu1, revealed higher relative cellular content of labile Cu in A2780.CisR cells compared with A2780 cells. These results demonstrate that Cu trafficking, homeostasis and speciation are different in the Pt-sensitive and resistant cells and may be associated with the predominance of different phenotypes for A2780 (epithelial) and A2780.CisR (mesenchymal) cells.

Sex-related differences in oxaliplatin-induced changes in the expression of transient receptor potential channels and their contribution to cold hypersensitivity.

Transient receptor potential (TRP) channels are involved in the development of oxaliplatin-induced neuropathic pain, a frequent and debilitating side effect of cancer therapy. Here we explored whether oxaliplatin-induced changes in the expression of TRP channels, as well as the development of pain-related behaviours, differed between male and female animals. Adult rats were injected with oxaliplatin or saline and mechanical and cold allodynia were evaluated using Von Frey and Choi Tests. The mRNA levels of TRPV1, TRPM8 and TRPA1 were assessed in lumbar ganglia and spinal cord by using real time RT-PCR. Oxaliplatin administration induced mechanical and cold hypersensitivity and allodynia in both sexes, with more severe responses to cold stimulation detected in females. Oxaliplatin also induced a significant increase in the expression of TRPV1, TRPM8 and TRPA1 in lumbar dorsal root ganglia. Interestingly, while TRPV1 and TRPA1 upregulation showed no sex difference, the increase in TRPM8 mRNA levels was more pronounced in female ganglia, correlating with the increased sensitivity to innocuous cold stimuli observed in females. TRPV1 and TRPM8 were also found to be upregulated in the spinal cord of animals of both sexes. Our results reveal previously undescribed changes in the expression of TRP channels occurring in peripheral ganglia and spinal cord of both male and female oxaliplatin-treated animals, with some of these changes exhibiting sex-related differences that could underlie the development of sex-specific patterns of pain-related behaviours.

Influence of Ring Modifications on Nucleolar Stress Caused by Oxaliplatin-Like Compounds.

Oxaliplatin, a platinum compound in broad clinical use, can induce cell death through a nucleolar stress pathway rather than the canonical DNA damage response studied for other Pt(II) compounds. Previous work has found that the oxaliplatin 1,2-diaminocyclohexane (DACH) ring but not the oxalate leaving group is important to the ability to induce nucleolar stress. Here we study the influence of DACH ring substituents at the 4-position on the ability of DACH-Pt(II) compounds to cause nucleolar stress. We determine that DACH-Pt(II) compounds with 4-position methyl, ethyl, or propyl substituents induce nucleolar stress, but DACH-Pt(II) compounds with 4-isopropyl substituents do not induce nucleolar stress. This effect is independent of whether the substituent is in the axial or equatorial position relative to the trans diamines of the ligand. These results suggest that spatially sensitive interactions could be involved in the ability of platinum compounds to cause nucleolar stress.

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.

Anticancer Activity, DNA Binding, and Photodynamic Properties of a N∧C∧N-Coordinated Pt(II) Complex.

In the effort to discover new targets and improve the therapeutic efficacy of metal-containing anticancer compounds, transition metal complexes that can elicit cytotoxicity when irradiated with light of a proper wavelength and, then, candidates as potential photosensitizers for photodynamic therapy are actively being investigated. In this work, the cytotoxicity in the dark and the photophysical properties of the complex Pt(N∧C∧N)Cl, where the N∧C∧N ligand is 2,6-dipyrido-4-methyl-benzene chloride, are investigated in detail by means of a series of theoretical levels, that is density functional theory and its time-dependent extension together with molecular dynamics (MD) simulations. In the dark, cytotoxicity has been explored by simulating the steps of the mechanism of action of classical Pt(II) complexes. The suitability of the investigated complex to act as a photosensitizer has been verified by calculating spectroscopic properties for both the unperturbed complex and its aquated and guanine-bound forms. Furthermore, using MD simulation outcomes as a starting point, the photophysical properties of DNA-intercalated and -bound complexes have been evaluated with the goal of establishing how intercalation and binding affect sensitization activity.

Platinum(II) N-heterocyclic carbene complexes arrest metastatic tumor growth.

Vimentin is a cytoskeletal intermediate filament protein that plays pivotal roles in tumor initiation, progression, and metastasis, and its overexpression in aggressive cancers predicted poor prognosis. Herein described is a highly effective antitumor and antimetastatic metal complex [PtII(C^N^N)(NHC2Bu)]PF6 (Pt1a; HC^N^N = 6-phenyl-2,2'-bipyridine; NHC= N-heterocyclic carbene) that engages vimentin via noncovalent binding interactions with a distinct orthogonal structural scaffold. Pt1a displays vimentin-binding affinity with a dissociation constant of 1.06 µM from surface plasmon resonance measurements and fits into a pocket between the coiled coils of the rod domain of vimentin with multiple hydrophobic interactions. It engages vimentin in cellulo, disrupts vimentin cytoskeleton, reduces vimentin expression in tumors, suppresses xenograft growth and metastasis in different mouse models, and is well tolerated, attributable to biotransformation to less toxic and renal-clearable platinum(II) species. Our studies uncovered the practical therapeutic potential of platinum(II)‒NHC complexes as effective targeted chemotherapy for combating metastatic and cisplatin-resistant cancers.

Investigating the anticancer properties of the two new platinum complexes with iso- and tert-pentylglycine by the DFT, molecular docking, and ADMET assessment and experimental confirmations.

In this study, two new anticancer platinum complexes formulated as [Pt(bpy)(L)]NO3 were synthesized using the iso and tert-pentylglycine ligands, two structural isomer ligands, to investigate side branches effect on the complex-DNA interaction. According to the comparative results of the ADMET assessment, these compounds can be considered as the drug-like molecules and oral medication. Mechanism of tumor inhibition and DNA binding parameters indicated the higher ability of the tert-isomer and also, both complexes acted through the disruption of the base pairs and stacks of helicity by the endothermic process. Fluorescence spectroscopy showed that the quenching mechanism is static for both drugs with large binding constant and high binding affinity towards the DNA. Also, the amount of binding constant of the tert -isomer was about 14 times of another structural isomerous complex. CD spectra indicated the conversion of the B-DNA into A-DNA form via electrostatic interaction for positively charged complexes. The cytotoxic data showed that both compounds have antiproliferative effects against the MCF-7 cell line and the inhibitory effect of the iso-derivative was better than the tert-one. Docking studies showed that the desolvation energy and hydrogen bond are more effective between the other interactions. The torsional free energy for both complexes mainly provided the groove binding along with partially electrostatic and intercalate binding. According to the density-functional theory data and because of positive electron density on the surface of complexes and facilitating of the metal drug to DNA phosphate groups approaching, both complexes may be good candidates for the anticancer drugs. Two new anticancer Pt(II) complexes were synthesized with glycine derivatives. In vitro cytotoxicity effects were tested against the human breast cancer cell line of MCF-7. Moreover, the modes of DNA binding with synthesized compounds were investigated using ADME prediction, DFT, molecular docking and spectroscopic methods.

Novel polyamide amidine anthraquinone platinum(II) complexes: cytotoxicity, cellular accumulation, and fluorescence distributions in 2D and 3D cell culture models.

1- and 1,5-Aminoalkylamine substituted anthraquinones (AAQs, 1C3 and 1,5C3) were peptide coupled to 1-, 2-, and 3-pyrrole lexitropsins to generate compounds that incorporated both DNA minor groove and intercalating moieties. The corresponding platinum(II) amidine complexes were synthesized through a synthetically facile amine-to-platinum mediated nitrile 'Click' reaction. The precursors as well as the corresponding platinum(II) complexes were biologically evaluated in 2D monolayer cells and 3D tumour cell models. Despite having cellular accumulation levels that were up to five-fold lower than that of cisplatin, the platinum complexes had cytotoxicities that were only three-fold lower. Accumulation was lowest for the complexes with two or three pyrrole groups, but the latter was the most active of the complexes exceeding the activity of cisplatin in the MDA-MB-231 cell line. All compounds showed moderate to good penetration into spheroids of DLD-1 cells with the distributions being consistent with active uptake of the pyrrole containing complexes in regions of the spheroids starved of nutrients.

Two new monofunctional platinum(II) dithiocarbamate complexes: phenanthriplatin-type axial protection, equatorial-axial conformational isomerism, and anticancer and DNA binding studies.

The syntheses of two platinum(ii) dithiocarbamate complexes (1 and 2) that show quinoplatin- and phenanthriplatin-type axial protection of the Pt-plane are described. The Pt-plane of complex 2 is axially more protected than that of complex 1. Furthermore, both complexes adopt two different stereochemical conformations in the solid state (based on single-crystal X-ray structures) owing to the structurally flexible piperazine backbone; i.e., C-e,e-Anti (1) and C-e,a-Syn (2), where "C" stands for the chair configuration, "e" and "a" stand for the equatorial and axial positions and "Anti" (opposite side) and "Syn" (same side) represent the relative orientations in space of the terminal substituents on the piperazine ring. In complex 2, the C-e,a-Syn conformation may provide additional steric hindrance to the Pt-plane. Despite the lower lipophilicity of 2 as compared to that of 1, the in vitro anticancer action against selected cancer cell lines is better for the former revealing the superior role of the axial protection over lipophilicity in modulating anticancer activity. The activity against the cancer promoting protein NF-κB signifies that the mode of cancer cell death may be the result of hindering the activity of NF-κB in the initiation of apoptosis. The apoptotic mode of cell death has been established earlier in a study using Annexin V-FITC. Finally, DNA binding studies revealed that the complex-DNA adduct formation is spontaneous and the mode of interaction is non-intercalative (electrostatic/covalent).

The effect of charge on the uptake and resistance to reduction of platinum(IV) complexes in human serum and whole blood models.

cis- and trans-Platinum(iv) complexes with diaminetetracarboxylate coordination spheres possess the highly desirable property of exhibiting unusual resistance to reduction by blood serum components and endogenous reductants such as ascorbate. At the same time they are rapidly reduced in the intracellular environment of cancer cells. Consequently, they can potentially be tuned to remain intact in vivo until arrival at the tumour target where they are rapidly reduced to yield the active platinum(ii) species. However, in order to achieve this, uptake must be largely restricted to tumour cells and therefore uptake by healthy cells including red blood cells must be prevented. In this proof of concept study, we report on the effect of net charge as a means of controlling the uptake by red blood cells. Using 1H NMR spectroscopy we found that modifying the net charge of the complex does not influence the rate of reduction of the complexes by an excess of ascorbate. Using XANES spectroscopy we found that modifying the net charge of the platinum(iv) complexes decreased the extent of reduction in whole blood, although probably not to the degree needed for the optimal delivery to tumours. Therefore, it is likely to be necessary to adopt higher charges and/or additional strategies to keep platinum(iv) prodrugs out of blood cells.

Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Unexpected photoactivation pathways in a folate-receptor-targeted trans-diazido Pt(IV) anticancer pro-drug.

A conjugate between a photoactive trans-diazido Pt(iv) pro-drug, trans,trans,trans-[Pt(N3)2(OH)2(py)2], and folic acid has been synthesized and fully characterized by high resolution ESI-MS, NMR and UV-vis spectroscopy. Photoactivation of the Pt-folate conjugate with visible light confirmed the generation of cytotoxic Pt(ii) species capable of binding to guanine nucleobases. Importantly, photoreduction of the Pt(iv) complex triggered the photodecomposition of the folate vector into a p-aminobenzoate-containing fragment and several pterin derivatives, including 6-formylpterin. Besides exhibiting high dark stability in physiological-like conditions, the Pt-folate conjugate was ca. 2× more photocytotoxic towards MCF-7 breast cancer cell line than its parent Pt(iv) complex with a high photoselectivity index (PI > 6.9). The higher photocytotoxicity of the conjugate may be a consequence of its higher cellular accumulation and of the generation of a set of different cytotoxic species, including Pt(ii) photoproducts and several pterin derivatives, which are known to generate ROS.

Insight into the antitumor actions of sterically hindered platinum(ii) complexes by a combination of STD NMR and LCMS techniques.

Sterically hindered platinum(ii) complexes have shown great advantages in overcoming platinum drug resistance. In this study, the antitumor actions of sterically hindered platinum(ii) complex 1 (cis-dichloro[(1R,2R)-N1-(2-fluorobenzyl)-1,2-diaminocyclohexane-N,N']platinum(ii), C13H19FPtCl2) were investigated by using saturation transfer difference nuclear magnetic resonance (STD NMR) and liquid chromatography-mass spectrometry (LCMS) techniques. STD NMR was applied to study the HSA (human serum albumin) binding properties, while the interactions between guanosine 5'-monophosphate (5'-GMP) and complex 1 were studied by LCMS. For HSA binding experiments, strong STD signals were observed for protons of sterically hindered parts of carrier ligands, indicating that the sterically hindered moieties of the carrier ligand could be situated inside the binding pocket of HSA. A 19F NMR experiment indicated that complex 1 could interact with HSA. Furthermore, the binding modes of complex 1 with guanosine 5'-monophosphate (5'-GMP) were studied in the absence and presence of glutathione by LCMS. According to the HPLC profiles, a mono-functional binding mode was observed for complex 1 both in the presence and in the absence of glutathione, while a bi-adduct was observed for Pt(DACH)Cl2, which may be one of the reasons for their different biological activities. Hence, this study demonstrated that the NMR method combined with the LCMS technique could provide valuable information to understand the transport and the underlying anticancer mechanisms of the platinum(ii) complex at the molecular level. Moreover, the results reported here can help to reveal the binding mechanisms of the sterically hindered platinum(ii) compounds with biomolecules, which may shed light on the design of novel platinum(ii) anticancer agents with suitable sterically hindered groups.

Anionic versus neutral Pt(II) complexes: The relevance of the charge for human serum albumin binding.

The focus of this work is pointing out the different behavior of two structurally related Pt(II) complexes, the anionic cyclometalated NBu4[(Bzq)Pt(Thio)], 1 and the neutral [(Phen)Pt(Thio)], 2, (Bzq = benzo[h]quinoline, Phen = 1,10-phenantroline, Thio = 1,2-benzenedithiolate), on the interaction with human serum albumin (HSA), a key drug-delivery protein in the bloodstream. Being very limited the number of anionic Pt(II) complexes reported to date, this is a pioneering example of report on a protein-ligand interaction involving a negatively charged platinum compound. The study was carried out by using fluorescence spectroscopy, differential scanning calorimetry and molecular docking simulations. The results revealed a strong binding affinity between the anionic compound and the protein, whereas a weak/moderate binding interaction was highlighted for the neutral one. Comparative studies with site specific ligands (warfarin and ibuprofen), allowed us to identify the protein binding sites of the two compounds. The work aims to shed light on the relevance of the charge in designing new drugs with a favorable binding affinity for HSA, which strongly contributes to influence their pharmacological and toxicological profile.

Metal complexes for mitochondrial bioimaging.

Mitochondria are essential organelles in eukaryotic cells, containing various signaling molecules and important enzymes associated with cell growth, death, and proliferation. The visualization of mitochondria and their biochemistry with confocal microscopy, fluorescence (phosphorescence) lifetime microscopy (FLIM, PLIM), and super-resolution microscopy has therefore been of great interest in recent years. In particular, transition metal complexes have emerged as excellent mitochondria-targeting probes, due to their high photostabilities, large Stokes shifts, tunable chemical structures and long luminescence lifetimes. In this review, we focus on platinum, ruthenium and iridium complexes, and their application as detectors of micro-environmental alterations as well as for the imaging of signaling molecules inside mitochondria.

Kinetic and Thermodynamic Investigation of Human Serum Albumin Interaction with Anticancer Glycine Derivative of Platinum Complex by Using Spectroscopic Methods and Molecular Docking.

In this paper, a new anticancer Pt (II) complex, cis-[Pt (NH3)2(tertpentylgly)]NO3, was synthesized with glycine-derivative ligand and characterized. Cytotoxicity of this water-soluble Pt complex was studied against human cancer breast cell line of MCF-7. The interaction of human serum albumin (HSA) with Pt complex was studied by using UV-Vis, fluorescence spectroscopy methods, and molecular docking at 27 and 37 °C in the physiological situation (I = 10 mM, pH = 7.4). The negative [Formula: see text] and positive [Formula: see text] indicated that electrostatic force may be a major mode in the binding between Pt complex and HSA. Binding constant values were obtained through UV-Vis and fluorescence spectroscopy that reveal strong interaction. The negative Gibbs free energy that was obtained by using the UV-Vis method offers spontaneous interaction. Fluorescence quenching the intensity of HSA by adding Pt complex confirms the static mode of interaction is effective for this binding process. Hill coefficients, nH, Hill constant, kH, complex aggregation number around HSA, , number of binding sites, g, HSA melting temperature, Tm, and Stern-Volmer constant, kSV, were also obtained. The kinetics of the interaction was studied, which showed a second-order kinetic. The results of molecular docking demonstrate the position of binding of Pt complex on HSA is the site I in the subdomain IIA.