Multiaction Pt(IV) Prodrugs Releasing Cisplatin and Dasatinib Are Potent Anticancer and Anti-Invasive Agents Displaying Synergism between the Two Drugs.

Herein, we describe the general design, synthesis, characterization, and biological activity of new multitargeting Pt(IV) prodrugs that combine antitumor cisplatin and dasatinib, a potent inhibitor of Src kinase. These prodrugs exhibit impressive antiproliferative and anti-invasive activities in tumor cell lines in both two-dimensional (2D) monolayers of cell cultures and three-dimensional (3D) spheroids. We show that the cisplatin moiety and dasatinib in the investigated Pt(IV) complexes are both involved in the mechanism of action in MCF7 breast cancer cells and act synergistically. Thus, combining dasatinib and cisplatin into one molecule, compared to using individual components in a mix, may bring several advantages, such as significantly higher activity in cancer cell lines and higher selectivity for tumor cells. Most importantly, Pt(IV)-dasatinib complexes hold significant promise for potential anticancer therapies by targeting epithelial-mesenchymal transition, thus preventing the spread and metastasis of tumors, a value unachievable by a simple combination of both individual components.

Novel 2-(5-Arylthiophen-2-yl)-benzoazole Cyclometalated Iridium(III) dppz Complexes Exhibit Selective Phototoxicity in Cancer Cells by Lysosomal Damage and Oncosis.

A second-generation series of biscyclometalated 2-(5-aryl-thienyl)-benzimidazole and -benzothiazole Ir(III) dppz complexes [Ir(C^N)2(dppz)]+, Ir1-Ir4, were rationally designed and synthesized, where the aryl group attached to the thienyl ring was p-CF3C6H4 or p-Me2NC6H4. These new Ir(III) complexes were assessed as photosensitizers to explore the structure-activity correlations for their potential use in biocompatible anticancer photodynamic therapy. When irradiated with blue light, the complexes exhibited high selective potency across several cancer cell lines predisposed to photodynamic therapy; the benzothiazole derivatives (Ir1 and Ir2) were the best performers, Ir2 being also activatable with green or red light. Notably, when irradiated, the complexes induced leakage of lysosomal content into the cytoplasm of HeLa cancer cells and induced oncosis-like cell death. The capability of the new Ir complexes to photoinduce cell death in 3D HeLa spheroids has also been demonstrated. The investigated Ir complexes can also catalytically photo-oxidate NADH and photogenerate 1O2 and/or •OH in cell-free media.

N-Indolyl diiron vinyliminium complexes exhibit antiproliferative effects in cancer cells associated with disruption of mitochondrial homeostasis, ROS scavenging, and antioxidant activity.

The indole scaffold has been established as a key organic moiety for developing new drugs; on the other hand, a range of diiron bis-cyclopentadienyl complexes have recently emerged for their promising anticancer potential. Here, we report the synthesis of novel diiron complexes with an indole-functionalized vinyliminium ligand (2-5) and an indole-lacking analogue for comparative purposes (6), which were characterized by analytical and spectroscopic techniques. Complexes 2-6 are substantially stable in DMSO­d6 and DMEM-d solutions at 37 °C (8% average degradation after 48 h) and display a balanced hydrophilic/lipophilic behaviour (LogPow values in the range -0.32 to 0.47), associated with appreciable water solubility. The complexes display selective antiproliferative potency towards several cancer cells in monolayer cultures, mainly in the low micromolar range, with reduced toxicity towards noncancerous epithelial cells. Thus, the cytotoxicity of the complexes is comparable to or better than clinically used metallopharmaceutical cisplatin. Comparing the antiproliferative activity obtained for complexes containing different ligands, we confirmed the importance of the indolyl group in the mechanism of antiproliferative activity of these complexes. Cell-based mechanistic studies suggest that the investigated diiron vinyliminium complexes (DVCs) show cytostatic rather than cytotoxic effects and subsequently induce a population of cells to undergo apoptosis. Furthermore, the molecular mechanism of action involves interactions with mitochondrial DNA and proteins, the reactive oxygen species (ROS)-scavenging properties and antioxidant activity of these complexes in cancer cells. This study highlights the importance of DVCs to their cancer cell activity and reinforces their prospective therapeutic potential as anticancer agents.

Multitargeting Prodrugs that Release Oxaliplatin, Doxorubicin and Gemcitabine are Potent Inhibitors of Tumor Growth and Effective Inducers of Immunogenic Cell Death.

A multitargeting prodrug (2) that releases gemcitabine, oxaliplatin, and doxorubicin in their active form in cancer cells is a potent cytotoxic agent with nM IC50s ; it is highly selective to cancer cells with mean selectivity indices to human (136) and murine (320) cancer cells. It effectively induces release of DAMPs (CALR, ATP & HMGB1) in CT26 cells facilitating more efficient phagocytosis by J774 macrophages than the FDA drugs or their co-administration. The viability of CT26 cells co-cultured with J774 macrophages and treated with 2 was reduced by 32 % compared to the non-treated cells, suggesting a synergistic antiproliferative effect between the chemical and immune reactions. 2 inhibited in vivo tumor growth in two murine models (LLC and CT26) better than the FDA drugs or their co-administration with significantly lower body weight loss. Mice inoculated with CT26 cells treated with 2 showed slightly better tumor free survival than doxorubicin.

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.

Platinum(IV) Derivatives of [Pt(1S,2S-diaminocyclohexane)(5,6-dimethyl-1,10-phenanthroline)] with Diclofenac Ligands in the Axial Positions: A New Class of Potent Multi-action Agents Exhibiting Selectivity to Cancer Cells.

The platinum(II) complex [Pt(1S,2S-diaminocyclohexane)(5,6-dimethyl-1,10-phenanthroline)]2+ (PtII56MeSS, 1) exhibits high potency across numerous cancer cell lines acting by a multimodal mechanism. However, 1 also displays side toxicity and in vivo activity; all details of its mechanism of action are not entirely clear. Here, we describe the synthesis and biological properties of new platinum(IV) prodrugs that combine 1 with one or two axially coordinated molecules of diclofenac (DCF), a non-steroidal anti-inflammatory cancer-selective drug. The results suggest that these Pt(IV) complexes exhibit mechanisms of action typical for Pt(II) complex 1 and DCF, simultaneously. The presence of DCF ligand(s) in the Pt(IV) complexes promotes the antiproliferative activity and selectivity of 1 by inhibiting lactate transporters, resulting in blockage of the glycolytic process and impairment of mitochondrial potential. Additionally, the investigated Pt(IV) complexes selectively induce cell death in cancer cells, and the Pt(IV) complexes containing DCF ligands induce hallmarks of immunogenic cell death in cancer cells.

Metallohelices stabilize DNA three-way junctions and induce DNA damage in cancer cells.

DNA three-way junctions (3WJ) represent one of the simplest supramolecular DNA structures arising as intermediates in homologous recombination in the absence of replication. They are also formed transiently during DNA replication. Here we examine the ability of Fe(II)-based metallohelices to act as DNA 3WJ binders and induce DNA damage in cells. We investigated the interaction of eight pairs of enantiomerically pure Fe(II) metallohelices with four different DNA junctions using biophysical and molecular biology methods. The results show that the metallohelices stabilize all types of tested DNA junctions, with the highest selectivity for the Y-shaped 3WJ and minimal selectivity for the 4WJ. The potential of the best stabilizer of DNA junctions and, at the same time, the most selective 3WJ binder investigated in this work to induce DNA damage was determined in human colon cancer HCT116 cells. These metallohelices proved to be efficient in killing cancer cells and triggering DNA damage that could yield therapeutic benefits.

Cyclometalated Benzimidazole Osmium(II) Complexes with Antiproliferative Activity in Cancer Cells Disrupt Calcium Homeostasis.

We present the synthesis and characterization of six new heteroleptic osmium(II) complexes of the type [Os(C^N)(N^N)2]OTf (N^N = 2,2'-bipyridine and dipyrido[3,2-d:2',3'-f]quinoxaline; C^N = deprotonated methyl 1-butyl-2aryl-benzimidazolecarboxylate) with varying substituents in the R3 position of the phenyl ring of the cyclometalating C^N ligand. The new compounds are highly kinetically inert and absorb a full-wavelength range of visible light. An investigation of the antiproliferative activity of the new compounds has been performed using a panel of human cancer and noncancerous 2D cell monolayer cultures under dark conditions and green light irradiation. The results demonstrate that the new Os(II) complexes are markedly more potent than conventional cisplatin. The promising antiproliferative activity of selected Os(II) complexes was also confirmed using 3D multicellular tumor spheroids, which have the characteristics of solid tumors and can mimic the tumor tissue microenvironment. The mechanism of antiproliferative action of complexes has also been investigated and revealed that the investigated Os(II) complexes activate the endoplasmic reticulum stress pathway in cancer cells and disrupt calcium homeostasis.

Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells.

Some metallo-supramolecular helical assemblies with size, shape, charge and amphipathic architectures similar to short cationic α-helical peptides have been shown to target and stabilise DNA G-quadruplexes (G4s) in vitro and downregulate the expression of G4-regulated genes in human cells. To expand the library of metallohelical structures that can act as efficient DNA G4 binders and downregulate genes containing G4-forming sequences in their promoter regions, we investigated the interaction of the two enantiomeric pairs of asymmetric Fe(II) triplex metallohelices with a series of five different DNA G4s formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC, c-KIT, and k-RAS oncogenes. The metallohelices display preferential binding to G4s over duplex DNA in all investigated G4-forming sequences and induced arrest of DNA polymerase on template strands containing G4-forming sequences. Moreover, the investigated metallohelices suppressed the expression of c-MYC and k-RAS genes at mRNA and protein levels in HCT116 human cancer cells, as revealed by RT-qPCR analysis and western blotting.

Is antitumor Pt(IV) complex containing two axial lonidamine ligands a true dual- or multi-action prodrug?

This work studied the mechanism of action of a Pt(IV) complex 2 bearing two axial lonidamine ligands, which are selective inhibitors of aerobic glycolysis. The presence of two lonidamine ligands in 2 compared to the parent Pt(II) complex increased its antiproliferative activity, cellular accumulation, and changed its cell cycle profile and mechanism of cell death. In 3D cell culture, 2 showed exceptional antiproliferative activity with IC50 values as low as 1.6 µM in MCF7 cells. The study on the influence of the lonidamine ligands in the Pt complex on glycolysis showed only low potency of ligands to affect metabolic processes in cancer cells, making the investigated complex, not a dual- or multi-action prodrug. However, the Pt(IV) prodrug effectively delivers the cytotoxic Pt(II) complex into cancer cells.

Gallium(III) Complex with Cloxyquin Ligands Induces Ferroptosis in Cancer Cells and Is a Potent Agent against Both Differentiated and Tumorigenic Cancer Stem Rhabdomyosarcoma Cells.

In this work, gallium(III) complex with cloxyquin (5-chloro-8-quinolinol, HClQ) ligands is shown to effectively inhibit proliferation of rhabdomyosarcoma cells, the frequent, aggressive, and poorly treatable cancer of children. It offers striking selectivity to cancer cells compared to noncancerous human fibroblasts. The data reveal that the complex induces ferroptosis in rhabdomyosarcoma cells, likely due to interfering with iron metabolism. Importantly, it can kill both bulk and stem rhabdomyosarcoma cells. To the best of our knowledge, this is the first compound based on metal other than Fe capable of inducing ferroptosis in cancer cells.

Novel cis-Pt(II) Complexes with Alkylpyrazole Ligands: Synthesis, Characterization, and Unusual Mode of Anticancer Action.

One concept of improving anticancer effects of conventional platinum-based antitumor drugs consists of conjugating these compounds with other biologically (antitumor) active agents, acting by a different mechanism. Here, we present synthesis, physicochemical characterization, biological effects, and mechanisms of action of four new analogs of conventional cisplatin, namely, cis-Pt(II) complexes containing either methyl or ethyl pyrazole N-donor ligands and chlorido or iodido ligands. It is noteworthy that while chlorido complexes display activity in a variety of cancer cell lines comparable to cisplatin, iodido complexes are considerably more potent due to their enhanced hydrophobicity and consequently enhanced cellular accumulation. Moreover, all of the studied Pt(II) alkylpyrazole complexes display a higher selectivity for tumor cells and effectively overcome the acquired resistance to cisplatin. Further results focused on the mechanism of action of the studied complexes and showed that in contrast to cisplatin and several platinum-based antitumor drugs, DNA damage by the investigated Pt(II)-alkylpyrazole complexes does not play a major role in their mechanism of action. Our findings demonstrate that inhibition of the tubulin kinesin Eg5, which is essential for forming a functional mitotic spindle, plays an important role in their mechanism of antiproliferative action.

Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles.

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

A [Pt(cis-1,3-diaminocycloalkane)Cl2] analog exhibits hallmarks typical of immunogenic cell death inducers in model cancer cells.

The platinum drugs belong to prevailing chemotherapeutics used in the treatment of cancer. At present, however, the search for new anticancer metal-based drugs that operate by the mechanisms distinct from those of the conventional chemotherapeutics is very active. Furthermore, it has been demonstrated that cytotoxic chemotherapy and immunotherapy may exert a highly synergistic anticancer activity. Thus, the development of antitumor platinum and other metal-based drugs that exhibit cytostatic effects and concurrently elicit immunogenic cell death (ICD) has shown promise for cancer treatment. Notably, conventional platinum drug oxaliplatin ([Pt(1R,2R-DACH)(oxalate)], DACH = diaminocyclohexane) is a well-known agent that displays both cytostatic and immune responses. Moreover, it was also demonstrated that even minor derivatization of the unleaving cycloalkyl moiety in oxaliplatin might have a pronounced effect on its immunomodulatory activity. Here, we investigated how replacing the 1R,2R- diaminocyclohexane ring by 1,3-diaminocycloalkane (alkane = butane, pentane, or hexane) affects the ability to evoke secretion of damage-associated molecular patterns characteristic of ICD in model murine colorectal carcinoma cell line CT26. The results indicate that among the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes, the complex containing the cyclobutyl moiety exhibits the hallmarks typical of ICD inducers. Thus, [Pt(cis-1,3-diaminocyclobutane)Cl2] may expand the spectrum of anticancer chemotherapeutics capable of inducing ICD in cancer cells and might be of interest for further (pre)clinical development.

Are Pt(IV) Prodrugs That Release Combretastatin A4 True Multi-action Prodrugs?

"Multi-action" Pt(IV) derivatives of cisplatin with combretastatin A4 (CA4) bioactive ligands that are conjugated to Pt(IV) by carbonate are unique because the ligand (IC50 < 10 nM) is dramatically 1000-folds more cytotoxic than cisplatin in vitro. The Pt(IV)-CA4 prodrugs were as cytotoxic as CA4 itself, indicating that the platinum moiety probably plays an insignificant role in triggering cytotoxicity, suggesting that the Pt(IV)-CA4 complexes act as prodrugs for CA4 rather than as true multi-action prodrugs. In vivo tests (Lewis lung carcinoma) show that ctc-[Pt(NH3)2(PhB)(CA4)Cl2] inhibited tumor growth by 93% compared to CA4 (67%), cisplatin (84%), and 1:1:1 cisplatin/CA4/PhB (85%) while displaying <5% body weight loss compared to cisplatin (20%) or CA4 (10%). In this case, and perhaps with other extremely potent bioactive ligands, platinum(IV) acts merely as a self-immolative carrier triggered by reduction in the cancer cell with only a minor contribution to cytotoxicity.

Hetero-Bis-Conjugation of Bioactive Molecules to Half-Sandwich Ruthenium(II) and Iridium(III) Complexes Provides Synergic Effects in Cancer Cell Cytotoxicity.

Four bipyridine-type ligands variably derivatized with two bioactive groups (taken from ethacrynic acid, flurbiprofen, biotin, and benzylpenicillin) were prepared via sequential esterification steps from commercial 2,2'-bipyridine-4,4'-dicarboxylic acid and subsequently coordinated to ruthenium(II) p-cymene and iridium(III) pentamethylcyclopentadienyl scaffolds. The resulting complexes were isolated as nitrate salts in high yields and fully characterized by analytical and spectroscopic methods. NMR and MS studies in aqueous solution and in cell culture medium highlighted a substantial stability of ligand coordination and a slow release of the bioactive fragments in the latter case. The complexes were assessed for their antiproliferative activity on four cancer cell lines, showing cytotoxicity to the low micromolar level (equipotent with cisplatin). Additional biological experiments revealed a multimodal mechanism of action of the investigated compounds, involving DNA metalation and enzyme inhibition. Synergic effects provided by specific combinations of metal and bioactive fragments were identified, pointing toward an optimal ethacrynic acid/flurbiprofen combination for both Ru(II) and Ir(III) complexes.

FeII Metallohelices Stabilize DNA G-Quadruplexes and Downregulate the Expression of G-Quadruplex-Regulated Oncogenes.

DNA G-quadruplexes (G4s) have been identified within the promoter regions of many proto-oncogenes. Thus, G4s represent attractive targets for cancer therapy, and the design and development of new drugs as G4 binders is a very active field of medicinal chemistry. Here, molecular biophysics and biology methods were employed to investigate the interaction of chiral metallohelices with a series of four DNA G4s (hTelo, c-myc, c-kit1, c-kit2) that are formed by the human telomeric sequence (hTelo) and in the promoter regions of c-MYC and c-KIT proto-oncogenes. We show that the investigated water-compatible, optically pure metallohelices, which are made by self-assembly of simple nonpeptidic organic components around FeII ions and exhibit bioactivity emulating the natural systems, bind with high affinity to G4 DNA and much lower affinity to duplex DNA. Notably, both enantiomers of a metallohelix containing a m-xylenyl bridge (5 b) were found to effectively inhibit primer elongation catalyzed by Taq DNA polymerase by stabilizing G4 structures formed in the template strands containing c-myc and c-kit2 G4-forming sequences. Moreover, both enantiomers of 5 b downregulated the expression of c-MYC and c-KIT oncogenes in human embryonic kidney cells at mRNA and protein levels. As metallohelices also bind alternative nucleic acid structures, they hold promise as potential multitargeted drugs.

A Cyclometalated IrIII Complex Conjugated to a Coumarin Derivative Is a Potent Photodynamic Agent against Prostate Differentiated and Tumorigenic Cancer Stem Cells.

A cyclometalated IrIII complex conjugated to a far-red-emitting coumarin, IrIII -COUPY (3), was recently shown as a very promising photosensitizer suitable for photodynamic therapy of cancer. Therefore, the primary goal of this work was to deepen knowledge on the mechanism of its photoactivated antitumor action so that this information could be used to propose a new class of compounds as drug candidates for curing very hardly treatable human tumors, such as androgen resistant prostatic tumors of metastatic origin. Conventional anticancer chemotherapies exhibit several disadvantages, such as limited efficiency to target cancer stem cells (CSCs), which are considered the main reason for chemotherapy resistance, relapse, and metastasis. Herein, we show, using DU145 tumor cells, taken as the model of hormone-refractory and aggressive prostate cancer cells resistant to conventional antineoplastic drugs, that the photoactivated conjugate 3 very efficiently eliminates both prostate bulk (differentiated) and prostate hardly treatable CSCs simultaneously and with a similar efficiency. Notably, the very low toxicity of IrIII -COUPY conjugate in the prostate DU145 cells in the dark and its pronounced selectivity for tumor cells compared with noncancerous cells could result in low side effects and reduced damage of healthy cells during the photoactivated therapy by this agent. Moreover, the experiments performed with the 3D spheroids formed from DU145 CSCs showed that conjugate 3 can penetrate the inner layers of tumor spheres, which might markedly increase its therapeutic effect. Also interestingly, this conjugate induces apoptotic cell death in prostate cancer DU145 cells associated with calcium signaling flux in these cells and autophagy. To the best of our knowledge, this is the first study demonstrating that a photoactivatable metal-based compound is an efficient agent capable of killing even hardly treatable CSCs.

Platinum(IV)-Estramustine Multiaction Prodrugs Are Effective Antiproliferative Agents against Prostate Cancer Cells.

Herein, we describe the synthesis, characterization, and biological properties of Pt(IV) derivatives of cisplatin with estramustine at the first axial position, which is known to disrupt the microtubule assembly and act as an androgen antagonist, and varying the second axial position using an innocent ligand (acetate or hydroxyl) to prepare dual-action and triple-action prodrugs with known inhibitors of histone deacetylase, cyclooxygenase, and pyruvate dehydrogenase kinase. We demonstrate superior antiproliferative activity at submicromolar concentrations of the prodrugs against a panel of cancer cell lines, particularly against prostate cancer cell lines. The results obtained in this study exemplify the complex mode of action of "multiaction" Pt(IV) prodrugs. Interestingly, changing the second axial ligand in the Pt-estramustine complex has a significant effect on the mode of action, suggesting that all three components of the Pt(IV) prodrugs (platinum moiety and axial ligands) contribute to the killing of cells and not just one dominant component.

A Multi-action PtIV Conjugate with Oleate and Cinnamate Ligands Targets Human Epithelial Growth Factor Receptor HER2 in Aggressive Breast Cancer Cells.

HER2-positive breast cancer is an aggressive subtype that typically responds poorly to standard chemotherapy. To design an anticancer drug selective for HER2-expressing breast cancer, a PtIV prodrug with axial oleate and cinnamate ligands was synthesized. We demonstrate its superior antiproliferative activity in monolayer and 3D spheroid models; the antiproliferative efficiency increases gradually with increasing expression of HER2. The results also suggest that the released PtII compound inhibits the proliferation of cancer cells by a DNA-damage-mediated mechanism. Simultaneously, the released oleic and cinnamic acid can effectively inhibit HER2 expression. To our knowledge, this is the first platinum-based complex inhibiting HER2 expression that does not contain protein or peptide. Moreover, this PtIV prodrug is capable of overcoming the resistance of cancer stem cells (CSCs), inducing death in both CSCs and differentiated cancer cells. Thus, the results substantiate our design strategy and demonstrate the potential of this approach for the development of new, therapeutically relevant compounds.