👤 Matés JM

Title: Anticancer activity of 8-hydroxyquinoline-triphenylphosphine rhodium(III) complexes targeting mitophagy pathways. Abstract: Metallodrugs exhibiting distinct mechanisms of action compared with cisplatin hold promise for overcoming cisplatin resistance and improving the efficacy of anticancer drugs. In this study, a new series of rhodium (Rh)(III) complexes containing tris(triphenylphosphine)rhodium(I) chloride [(TPP)3RhCl] (TPP = triphenylphosphine, TPP=O = triphenylphosphine oxide) and 8-hydroxyquinoline derivatives (H-XR1-H-XR4), namely [Rh(XR1)2(TPP)Cl]·(TPP=O) (Yulin Normal University-1a [YNU-1a]), [Rh(XR2)2(TPP)Cl] (YNU-1b), [Rh(XR3)2(TPP)Cl] (YNU-1c), and [Rh(XR4)2(TPP)Cl] (YNU-1d), was synthesized and characterized via X-ray diffraction, mass spectrometry and IR. The cytotoxicity of the compounds YNU-1a-YNU-1d in Hep-G2 and HCC1806 human cancer cell lines and normal HL-7702 cell line was evaluated. YNU-1c exhibited cytotoxicity and selectivity in HCC1806 cells (IC50 = 0.13 ± 0.06 μM, selectivity factor (SF) = 384.6). The compounds YNU-1b and YNU-1c, which were selected for mechanistic studies, induced the activation of apoptotic pathways and mitophagy. In addition, these compounds released cytochrome c, cleaved caspase-3/pro-caspase-3 and downregulated the levels of mitochondrial respiratory chain complexes I/IV (M1 and M4) and ATP. The compound YNU-1c, which was selected for in vivo experiments, exhibited tumor growth inhibition (58.9 %). Importantly, hematoxylin and eosin staining and TUNEL revealed that HCC1806 tumor tissues exhibited significant apoptotic characteristics. YNU-1a-YNU-1d compounds are promising drug candidates that can be used to overcome cisplatin resistance. Show less

Natural products represent an excellent source of unprecedented anticancer compounds. However, the identification of the mechanism of action remains a major challenge. Several techniques and methodologies have been considered, but with limited success. In this work, we explored the combination of live cell imaging and machine learning techniques as a promising tool to depict in a fast and affordable test the mode of action of natural compounds with antiproliferative activity. To develop the model, we selected the non-small cell lung cancer cell line SW1573, which was exposed to the known antimitotic drugs paclitaxel, colchicine and vinblastine. The novelty of our methodology focuses on two main features with the highest relevance, (a) meaningful phenotypic metrics, and (b) fast Fourier transform (FFT) of the time series of the phenotypic parameters into their corresponding amplitudes and phases. The resulting algorithm was able to cluster the microtubule disruptors, and meanwhile showed a negative correlation between paclitaxel and the other treatments. The FFT approach was able to group the samples as efficiently as checking by eye. This methodology could easily scale to group a large amount of data without visual supervision. Show less

CCAAT/enhancer binding protein β (C/EBPβ) is a basic leucine zipper (bZIP) family transcription factor, which is upregulated or overactivated in many cancers, resulting in a gene expression profile that drives oncogenesis. C/EBPβ dimerization regulates binding to DNA at the canonical TTGCGCAA motif and subsequent transcriptional activity, suggesting that disruption of dimerization represents a powerful approach to inhibit this previously "undruggable" oncogenic target. Here we describe the mechanism of action and antitumor activity of ST101, a novel and selective peptide antagonist of C/EBPβ that is currently in clinical evaluation in patients with advanced solid tumors. ST101 binds the leucine zipper domain of C/EBPβ, preventing its dimerization and enhancing ubiquitin-proteasome dependent C/EBPβ degradation. ST101 exposure attenuates transcription of C/EBPβ target genes, including a significant decrease in expression of survival, transcription factors, and cell-cycle-related proteins. The result of ST101 exposure is potent, tumor-specific in vitro cytotoxic activity in cancer cell lines including glioblastoma, breast, melanoma, prostate, and lung cancer, whereas normal human immune and epithelial cells are not impacted. Further, in mouse xenograft models ST101 exposure results in potent tumor growth inhibition or regression, both as a single agent and in combination studies. These data provide the First Disclosure of ST101, and support continued clinical development of ST101 as a novel strategy for targeting C/EBPβ-dependent cancers. Show less

Stimulation of plasma membrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), locally increases the abundance of reactive oxygen species (ROS). These ROS then oxidize cysteine residues in proteins to potentiate downstream signaling. Spatial confinement of ROS is an important regulatory mechanism of redox signaling that enables the stimulation of different RTKs to oxidize distinct sets of downstream proteins. To uncover additional mechanisms that specify cysteines that are redox regulated by EGF stimulation, we performed time-resolved quantification of the EGF-dependent oxidation of 4200 cysteine sites in A431 cells. Fifty-one percent of cysteines were statistically significantly oxidized by EGF stimulation. Furthermore, EGF induced three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamics simulations indicated widespread redox regulation of cryptic cysteine residues that are solvent exposed only upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates served as two distinct modes by which EGF specified the cryptic cysteine residues that became solvent exposed and redox regulated. Because proteins that are structurally regulated by different RTKs or cellular perturbations are largely unique, these findings suggest that solvent exposure and redox regulation of cryptic cysteine residues contextually delineate redox signaling networks. Show less

The study of cancer metabolism is regaining center stage and becoming a hot topic in tumor biology and clinical research, after a period where such kind of experimental approaches were somehow forgotten or disregarded in favor of powerful functional genomic and proteomic studies [...]. Show less

A family of six Ru(II) polypyridyl complexes (1-6) which contain phenanthroline-based ligands functionalized with alkyl chains of different lengths (one methyl group, 10 and 21 carbon alkyl chains) and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesized and characterized. The influence of the alkyl chain length on their photophysical and photochemical properties as well as in their photobiological applications has been elucidated by monitoring the changes in their MLCT-centered absorption and emission bands. The presence of one methyl group or 10 carbon alkyl chains does not seem to significantly affect the photophysical and photochemical properties of the resulting Ru(II) complexes when compared to the well-known [Ru(phen)₃]²⁺ and [Ru(TAP)₂phen]²⁺. However, an effect on their emission properties and in their ability to photosensitize singlet oxygen is observed for the Ru(II) complexes containing 21 carbon alkyl chains. The binding of these complexes to salmon testes DNA (stDNA) was investigated by observing the changes in the photophysical properties. Complexes 1, 2, 4, and 5 all showed changes in their MLCT bands that could be analyzed using conventional fitting methods, such as the Bard equation. In contrast, complexes 3 and 6, possessing long aliphatic chains, gave rise to nonclassic behavior. In addition to these analyses, both thermal denaturation and circular dichroism studies of 1-6 were carried out in the presence of stDNA which confirmed that these complexes bind to DNA. Confocal microscopy and viability studies in HeLa cervical cancer cells reveal an alkyl chain-length dependence on the cellular uptake and cytotoxicity of the resulting Ru(II) complexes due to an enhancement of their lipophilicity with increasing alkyl chain length. Thus, complexes containing 10 and 21 carbon alkyl chains are rapidly taken up into HeLa cells and, in particular, those with 21 carbon alkyl chains show a significant phototoxicity against the same cell line. Therefore, this study provides further insight into the possible modulation of the photophysical, photochemical, and photobiological properties of Ru(II) polypyridyl complexes by varying the length of the alkyl chains attached to the polypyridyl ligands coordinated to the Ru(II) center and the nature of the auxiliary groups, which we show has a significant effect on photophysical and biological properties. Show less

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents. Show less

The transcription factor NRF2 (nuclear factor-erythroid 2 p45-related factor 2 or NFE2L2) plays a critical role in response to cellular stress. Following an oxidative insult, NRF2 orchestrates an antioxidant program, leading to increased glutathione levels and decreased reactive oxygen species (ROS). Mounting evidence now implicates the ability of NRF2 to modulate metabolic processes, particularly those at the interface between antioxidant processes and cellular proliferation. Notably, NRF2 regulates the pentose phosphate pathway, NADPH production, glutaminolysis, lipid and amino acid metabolism, many of which are hijacked by cancer cells to promote proliferation and survival. Moreover, deregulation of metabolic processes in both normal and cancer-based physiology can stabilize NRF2. We will discuss how perturbation of metabolic pathways, including the tricarboxylic acid (TCA) cycle, glycolysis, and autophagy can lead to NRF2 stabilization, and how NRF2-regulated metabolism helps cells deal with these metabolic stresses. Finally, we will discuss how the negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), may play a role in metabolism through NRF2 transcription-independent mechanisms. Collectively, this review will address the interplay between the NRF2/KEAP1 complex and metabolic processes. Show less

Ninety-seven percent of drug-indication pairs that are tested in clinical trials in oncology never advance to receive U.S. Food and Drug Administration approval. While lack of efficacy and dose-limiting toxicities are the most common causes of trial failure, the reason(s) why so many new drugs encounter these problems is not well understood. Using CRISPR-Cas9 mutagenesis, we investigated a set of cancer drugs and drug targets in various stages of clinical testing. We show that-contrary to previous reports obtained predominantly with RNA interference and small-molecule inhibitors-the proteins ostensibly targeted by these drugs are nonessential for cancer cell proliferation. Moreover, the efficacy of each drug that we tested was unaffected by the loss of its putative target, indicating that these compounds kill cells via off-target effects. By applying a genetic target-deconvolution strategy, we found that the mischaracterized anticancer agent OTS964 is actually a potent inhibitor of the cyclin-dependent kinase CDK11 and that multiple cancer types are addicted to CDK11 expression. We suggest that stringent genetic validation of the mechanism of action of cancer drugs in the preclinical setting may decrease the number of therapies tested in human patients that fail to provide any clinical benefit. Show less

Two water-soluble amphiphilic Ru(ii) polypyridyl complexes containing N-1,10-phenanthrolin-5-yldocosanamide and 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ligands were synthesised and their photophysical and photobiological properties evaluated; both complexes showed a rapid cellular uptake and displayed phototoxicity against HeLa cervical cancer cells. Show less

The histone deacetylase (HDAC) enzymes have emerged as an important class of molecular targets in cancer therapy, with five inhibitors in clinical use. Recently, it has been shown that a lack of selectivity between the 11 Zn-dependent HDAC isoforms may lead to unwanted side-effects. In this paper, we show that piano stool Ru complexes can act as HDAC inhibitors, and variation in the capping arene leads to differences in HDAC isoform selectivity. Show less

We report cytotoxic ruthenium(ii) complexes of the general formula [RuCl(cis-tach)(diphosphine)]+ (cis-tach = cis-cis-1,3,5-triaminocyclohexane) that have been characterised by 1H, 13C and 31P{1H} NMR spectroscopy, mass spectrometry, X-ray crystallography and elemental analysis. The kinetics of aquation and stability of the active species have been studied, showing that the chlorido ligand is substituted by water at 298 K with first order rate constants of 10-2-10-3 s-1, ideal for potential clinical use as anti-tumour agents. Strong interactions with biologically relevant duplex and quadruplex DNA models correlate with the activity observed with A549, A2780 and 293T cell lines, and the degree of activity was found to be sensitive to the chelating diphosphine ligand. A label-free ptychographic cell imaging technique recorded cell death processes over 4 days. The Ru(ii) cis-tach diphosphine complexes exhibit anti-proliferative effects, in some cases outperforming cisplatin and other cytotoxic ruthenium complexes. Show less

Herein the design and synthesis of a new 8-hydroxyquinoline derivative, (S)-5-chloro-7-((proline-1-yl)methyl)8-hydroxyquinoline (HQCl-Pro), with good water solubility and multidrug resistance reversal activity are reported. In this work the proton dissociation processes of HQCl-Pro and its complex formation with [Rh(η⁵-C₅Me₅)(H₂O)₃]²⁺, [Ru(η⁶-p-cymene)(H₂O)₃]²⁺ and [Ru(η⁶-toluene)(H₂O)₃]²⁺ were investigated by the combined use of pH-potentiometry, UV-visible spectrometry and ¹H NMR spectroscopy. Our results revealed the prominent solution stability of the complexes in all cases. The lipophilicity of the complexes increased with the chloride ion concentration, and the complexes showed moderate log D values (-0.8 to +0.4) at pH 7.4 at all tested Cl^- concentrations. The formation of mixed hydroxido complexes from the aqua complexes was characterized by relatively high pK_a values (8.45-9.62 in chloride-free medium). Complexation processes are much slower with the Ru(η⁶-arene) triaqua cations than with [Rh(η⁵-C₅Me₅)(H₂O)₃]²⁺. Both the pK_a values and H₂O/Cl^- exchange constants of the Ru-complexes are lower by 0.5-1.0 orders of magnitude than those of the Rh analogue. Arene loss (p-cymene and toluene) and oxidation were found in the case of Ru-complexes when an excess of HQCl-Pro and aromatic (N,N) bidentate ligands was added. The cytotoxicity and antiproliferative effect of HQCl-Pro and its complexes were assayed in vitro. In contrast to the structurally familiar 8-hydroxyquinoline, HQCl-Pro and its Rh(η⁵-C₅Me₅) complex were somewhat more effective against drug resistant Colo 320 adenocarcinoma human cells compared to the drug sensitive Colo 205 cells. The Ru- and Rh-complexes showed a similar metal uptake level after 4 h, while a longer incubation time resulted in higher cellular Rh concentration. Show less

Mammalian tissues are fuelled by circulating nutrients, including glucose, amino acids, and various intermediary metabolites. Under aerobic conditions, glucose is generally assumed to be burned fully by tissues via the tricarboxylic acid cycle (TCA cycle) to carbon dioxide. Alternatively, glucose can be catabolized anaerobically via glycolysis to lactate, which is itself also a potential nutrient for tissues and tumours. The quantitative relevance of circulating lactate or other metabolic intermediates as fuels remains unclear. Here we systematically examine the fluxes of circulating metabolites in mice, and find that lactate can be a primary source of carbon for the TCA cycle and thus of energy. Intravenous infusions of 13C-labelled nutrients reveal that, on a molar basis, the circulatory turnover flux of lactate is the highest of all metabolites and exceeds that of glucose by 1.1-fold in fed mice and 2.5-fold in fasting mice; lactate is made primarily from glucose but also from other sources. In both fed and fasted mice, 13C-lactate extensively labels TCA cycle intermediates in all tissues. Quantitative analysis reveals that during the fasted state, the contribution of glucose to tissue TCA metabolism is primarily indirect (via circulating lactate) in all tissues except the brain. In genetically engineered lung and pancreatic cancer tumours in fasted mice, the contribution of circulating lactate to TCA cycle intermediates exceeds that of glucose, with glutamine making a larger contribution than lactate in pancreatic cancer. Thus, glycolysis and the TCA cycle are uncoupled at the level of lactate, which is a primary circulating TCA substrate in most tissues and tumours. Show less

A new family of neutral ruthenium(II) arene complexes of the type [Ru(η⁶-arene)X(κ²- O, N-L)] (η⁶-arene = p-cym, bz; X = Cl^-, SCN^-; HL1 = 2-(2'-hydroxyphenyl)benzimidazole, HL2 = 2-(2'-hydroxyphenyl)benzothiazole) has been synthesized and characterized. The cytotoxic activity of the Ru(II) complexes was evaluated in several tumor cell lines (A549, HepG2 and SW480) both in the dark and after soft irradiation with UV and blue light. None of the complexes bearing benzimidazole (HL1) as a ligand displayed phototoxicity, whereas the complexes with a benzothiazole ligand (HL2) exhibited photoactivation; the sensitivity observed for UV was higher than for blue light irradiation. The interesting results displayed by HL2 and [Ru(η⁶- p-cym)(NCS)(κ²- O, N-L2)], [3a], in terms of photo cytotoxicity prompted us to analyze their interaction with DNA, both in the dark and under irradiation conditions, in an effort to shed some light on their mechanism of action. The results of this study revealed that HL2 interacts with DNA by groove binding, whereas [3a] interacts by a dual mode of binding, an external groove binding, and covalent binding of the metal center to the guanine moiety. Interestingly, both HL2 and [3a] display a clear preference for AT base pairs, and this causes fluorescence enhancement. Additionally, cleavage of the pUC18 plasmid DNA by the complex is observed upon irradiation. The study of the irradiated form demonstrates that the arene ligand is released to yield species such as [Ru(κ²- O, N-L2)(κ¹- S-DMSO)₂(μ-SCN)]₂ [3c] and [Ru(κ²- O, N-L2)(κ¹- S-DMSO)₃(SCN)] [3d]. Such photo dissociation occurs even in the absence of oxygen and leads to cytotoxicity enhancement, an effect attributed to the presence of [3d], thus revealing the potential of [3a] as a pro-drug for photoactivated anticancer chemotherapy (PACT). Show less

Five Ru(II)(η⁶-toluene) complexes formed with 2-picolinic acid and its various derivatives have been synthesized and characterized. X-ray structures of four complexes are also reported. Complex formation processes of [Ru(II)(η⁶-toluene)(H₂O)₃]²⁺ organometallic cation with the metal-free ligands were studied in aqueous solution in the presence of chloride ions by the combined use of ¹H NMR spectroscopy, UV-visible spectrophotometry and pH-potentiometry. Solution stability, chloride ion affinity and lipophilicity of the complexes were characterized together with in vitro cytotoxic and antiproliferative activity in cancer cell lines being sensitive and resistant to classic chemotherapy and in normal cells as well. Formation of mono complexes such as [Ru(η⁶-toluene)(L)(Z)]^+/0 (L: completely deprotonated ligand; Z = H₂O/Cl^-) with high stability and [Ru(η⁶-toluene)(L)(OH)] was found in solution. The pK_a values (8.3-8.7) reflect the formation of low amount of mixed hydroxido species at pH 7.4 at 0.2 M KCl ionic strength. The complexes are fairly hydrophilic and show moderate chloride ion affinity and fast chloride-water exchange processes. The studied complexes exhibit no cytotoxic activity in human cancer cells (IC₅₀ > 100 μM), only complexes formed with 2-picolinic acid (1) and its 3-methyl derivative (2) represented a moderate antiproliferative effect (IC₅₀ = 84.8 (1), 79.2 μM (2)) on a multidrug resistant colon adenocarcinoma cell line revealing considerable multidrug resistant selectivity. Complexes 1 and 2 bind to human serum albumin covalently and relatively slowly with moderate strength at multiple binding sites without ligand cleavage. Show less

Considering the promising previous results of ct-[RuCl(CO)(dppb)(bipy)]PF₆ (where dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2'-bipyridine) as an antitumor agent, novel biological assays evaluating its toxicogenic potential were performed. The genotoxicity of the compound was evaluated by the in vitro micronucleus test (V79, Chinese hamster lung fibroblasts; HepG2, hepatocellular carcinoma cells), in vivo bone marrow micronucleus test and comet assay in hepatocytes (Swiss mice). The animals were treated with 0.63, 1.25, 2.5 and 5.0 mg/kg body weight (bw) of the compound. Negative (water) and positive (cisplatin, 1.5 mg/kg bw; methyl methanesulfonate, 40 mg/kg bw) controls were included. The parameters considered in the comet assay were the percentage of tail DNA, tail moment and tail length. The results of the in vitro micronucleus tests showed the absence of genotoxicity in V79 cells, while the compound was genotoxic in HepG2 cells at a concentration of 1.25 μm. In the in vivo micronucleus test, the compound was not genotoxic at the different doses evaluated. In the comet assay, only the dose of 5.0 mg/kg bw resulted in a significant increase in the frequency of DNA damage in hepatocytes when compared to the negative control. The genotoxic effect observed in HepG2 cells and in the liver comet assay indicates that the compound was metabolized by hepatic cells. Show less

A series of 2(1H)-quinolinone derivatives and their rhodium (III) complexes were designed and synthesized. All the rhodium (III) complexes exhibited higher in vitro cytotoxicity for Hep G2, HeLa 229, MGC80-3, and NCI-H460 human tumor cell lines than their ligands and cisplatin, and among them complex 9 was found to be selectively cytotoxic to tumor cells. Further investigation revealed that complex 9 caused cell cycle arrest at the G2/M phase and induced apoptosis, and inhibited the proliferation of Hep G2 cells by impeding the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream enzymes. Complex 9 also up-regulated the proapoptotic proteins Bak, Bax, and Bim, which altogether activated caspase-3/9 to initiate cell apoptosis. Notably, complex 9 effectively inhibited tumor growth in the NCI-H460 xenograft mouse model with less adverse effect than cisplatin. Show less

Oxidative stress is thought to account for aberrant redox homeostasis and contribute to aging and disease. However, more often than not, administration of antioxidants is ineffective, suggesting that our current understanding of the underlying regulatory processes is incomplete. Recent Advances: Similar to reactive oxygen species and reactive nitrogen species, reactive sulfur species are now emerging as important signaling molecules, targeting regulatory cysteine redox switches in proteins, affecting gene regulation, ion transport, intermediary metabolism, and mitochondrial function. To rationalize the complexity of chemical interactions of reactive species with themselves and their targets and help define their role in systemic metabolic control, we here introduce a novel integrative concept defined as the reactive species interactome (RSI). The RSI is a primeval multilevel redox regulatory system whose architecture, together with the physicochemical characteristics of its constituents, allows efficient sensing and rapid adaptation to environmental changes and various other stressors to enhance fitness and resilience at the local and whole-organism level. Show less

Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ∼400 000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap 'N' Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC. Show less

Seven rhenium(I) complexes of the general formula fac-[Re(CO)₃(NN)(OH₂)]⁺ where NN = 2,2'-bipyridine (8), 4,4'-dimethyl-2,2'-bipyridine (9), 4,4'-dimethoxy-2,2'-bipyridine (10), dimethyl 2,2'-bipyridine-4,4'-dicarboxylate (11), 1,10-phenanthroline (12), 2,9-dimethyl-1,10-phenanthroline (13), or 4,7-diphenyl-1,10-phenanthroline (14), were synthesized and characterized by ¹H NMR spectroscopy, IR spectroscopy, mass spectrometry, and X-ray crystallography. With the exception of 11, all complexes exhibited 50% growth inhibitory concentration (IC₅₀) values that were less than 20 μM in HeLa cells, indicating that these compounds represent a new potential class of anticancer agents. Complexes 9, 10, and 13 were as effective in cisplatin-resistant cells as wild-type cells, signifying that they circumvent cisplatin resistance. The mechanism of action of the most potent complex, 13, was explored further by leveraging its intrinsic luminescence properties to determine its intracellular localization. These studies indicated that 13 induces cytoplasmic vacuolization that is lysosomal in nature. Additional in vitro assays indicated that 13 induces cell death without causing an increase in intracellular reactive oxygen species or depolarization of the mitochondrial membrane potential. Further studies revealed that the mode of cell death does not fall into one of the canonical categories such as apoptosis, necrosis, paraptosis, and autophagy, suggesting that a novel mode of action may be operative for this class of rhenium compounds. The in vivo biodistribution and metabolism of complex 13 and its ^99mTc analogue 13* were also evaluated in naı̈ve mice. Complexes 13 and 13* exhibited comparable biodistribution profiles with both hepatic and renal excretion. High-performance liquid chromatography inductively coupled plasma mass-spectrometry (HPLC-ICP-MS) analysis of mouse blood plasma and urine postadministration showed considerable metabolic stability of 13, rendering this potent complex suitable for in vivo applications. These studies have shown the biological properties of this class of compounds and demonstrated their potential as promising theranostic anticancer agents that can circumvent cisplatin resistance. Show less

Four new Ru(ii) polypyridyl complexes that contain an extended aromatic moiety derived from pyrazino[2,3-h]dipyrido[3,2-a:2',3'-c]phenazine and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) have been synthesized, their solid state X-ray crystal structure determined and their photophysical and biological properties evaluated. Their interactions with DNA have been studied, and they have been tested for their potential as photodynamic therapeutic (PDT) agents in the treatment of cancer. A practical modification of a method by Carter, Rodriguez and Bard has been introduced and used to calculate binding parameters for the complexes which show a strong affinity for DNA with binding constants in the order of 10⁷ M^-1 (in 10 mM phosphate buffer). The complexes containing phen as an ancillary ligand become emissive upon binding to DNA ("light switch effect"), but do not show selective cytotoxicity upon light irradiation. On the other hand, the TAP complexes, which show an inverse "light switch effect" (emission quenched upon binding to DNA), are strongly photo-toxic suggesting their use in Photodynamic Therapy (PDT). In HeLa cells the best PDT agent shows an IC₅₀ value (light) = 4 μM vs. IC₅₀ value (dark) = 62 μM. Show less

The first examples of Ru^II and Rh^III piano-stool complex histone deacetylase (HDAC) inhibitors are presented. The novel complexes have antiproliferative activity against H460 non-small-cell lung carcinoma cells that is comparable to the clinically used HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Strong evidence for HDAC inhibition as a primary mechanism of action is provided. The complexes reported here represent an important step towards the design of highly active and selective HDAC inhibitors. Show less

A range of 1,4-substituted 2-pyridyl-N-phenyl triazoles were synthesised and evaluated for their antiproliferative properties against lymph node cancer of the prostate (LNCaP) and bone metastasis of prostate cancer (PC-3) cells. Excellent-to-low IC50 values were determined (5.6-250 μM), and a representative group of 4 ligands were then complexed to iridium(III) giving highly luminescent species. Re-evaluation of these compounds against both cell lines was then undertaken and improved potency (up to 72-fold) was observed, giving IC50 values of 0.36-11 μM for LNCaP and 0.85-5.9 μM for PC-3. Preliminary screens for in vivo toxicity were conducted using a zebrafish model showing a wide range of induced toxicity depending of the compound evaluated. Apoptosis and Caspase-3 levels were also determined and showed no statistical difference between some of the treated specimens and the controls. This study may identify novel therapeutic agents for advanced stage of prostate cancer in humans. Show less

The organometallic "half-sandwich" compound [Os(η(6)-p-cymene)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6 is 49× more potent than the clinical drug cisplatin in the 809 cancer cell lines that we screened and is a candidate drug for cancer therapy. We investigate the mechanism of action of compound 1 in A2780 epithelial ovarian cancer cells. Whole-transcriptome sequencing identified three missense mutations in the mitochondrial genome of this cell line, coding for ND5, a subunit of complex I (NADH dehydrogenase) in the electron transport chain. ND5 is a proton pump, helping to maintain the coupling gradient in mitochondria. The identified mutations correspond to known protein variants (p.I257V, p.N447S, and p.L517P), not reported previously in epithelial ovarian cancer. Time-series RNA sequencing suggested that osmium-exposed A2780 cells undergo a metabolic shunt from glycolysis to oxidative phosphorylation, where defective machinery, associated with mutations in complex I, could enhance activity. Downstream events, measured by time-series reverse-phase protein microarrays, high-content imaging, and flow cytometry, showed a dramatic increase in mitochondrially produced reactive oxygen species (ROS) and subsequent DNA damage with up-regulation of ATM, p53, and p21 proteins. In contrast to platinum drugs, exposure to this organo-osmium compound does not cause significant apoptosis within a 72-h period, highlighting a different mechanism of action. Superoxide production in ovarian, lung, colon, breast, and prostate cancer cells exposed to three other structurally related organo-Os(II) compounds correlated with their antiproliferative activity. DNA damage caused indirectly, through selective ROS generation, may provide a more targeted approach to cancer therapy and a concept for next-generation metal-based anticancer drugs that combat platinum resistance. Show less

Reactive oxygen species (ROS) are well known to be involved in oncogene-mediated cellular transformation. However, the regulatory mechanisms underlying ROS generation in oncogene-transformed cells are unclear. In the present study, we found that oncogenic K-Ras induces ROS generation through activation of NADPH oxidase 1 (NOX1), which is a critical regulator for the K-Ras-induced cellular transformation. NOX1 was activated by K-Ras-dependent translocation of p47(phox), a subunit of NOX1 to plasma membrane. Of note, PKCδ, when it was activated by PDPK1, directly bound to the SH3-N domain of p47(phox) and catalyzed the phosphorylation on Ser348 and Ser473 residues of p47(phox) C-terminal in a K-Ras-dependent manner, finally leading to its membrane translocation. Notably, oncogenic K-Ras activated all MAPKs (JNK, ERK and p38); however, only p38 was involved in p47(phox)-NOX1-dependent ROS generation and consequent transformation. Importantly, K-Ras-induced activation of p38 led to an activation of PDPK1, which then signals through PKCδ, p47(phox) and NOX1. In agreement with the mechanism, inhibition of p38, PDPK1, PKCδ, p47(phox) or NOX1 effectively blocked K-Ras-induced ROS generation, anchorage-independent colony formation and tumor formation. Taken together, our findings demonstrated that oncogenic K-Ras activates the signaling cascade p38/PDPK1/PKCδ/p47(phox)/NOX1 for ROS generation and consequent malignant cellular transformation. Show less

The ligands 2-pyridin-2-yl-1H-benzimidazole (HL(1)), 1-methyl-2-pyridin-2-ylbenzimidazole (HL(2)), and 2-(1H-imidazol-2-yl)pyridine (HL(3)) and the proligand 2-phenyl-1H-benzimidazole (HL(4)) have been used to prepare five different types of new ruthenium(II) arene compounds: (i) monocationic complexes with the general formula [(η(6)-arene)RuCl(κ(2)-N,N-HL)]Y [HL = HL(1), HL(2), or HL(3); Y = Cl or BF4; arene = 2-phenoxyethanol (phoxet), benzene (bz), or p-cymene (p-cym)]; (ii) dicationic aqua complexes of the formula [(η(6)-arene)Ru(OH2)(κ(2)-N,N-HL(1))](Y)2 (Y = Cl or TfO; arene = phoxet, bz, or p-cym); (iii) the nucleobase derivative [(η(6)-arene)Ru(9-MeG)(κ(2)-N,N-HL(1))](PF6)2 (9-MeG = 9-methylguanine); (iv) neutral complexes consistent with the formulation [(η(6)-arene)RuCl(κ(2)-N,N-L(1))] (arene = bz or p-cym); (v) the neutral cyclometalated complex [(η(6)-p-cym)RuCl(κ(2)-N,C-L(4))]. The cytototoxic activity of the new ruthenium(II) arene compounds has been evaluated in several cell lines (MCR-5, MCF-7, A2780, and A2780cis) in order to establish structure-activity relationships. Three of the compounds with the general formula [(η(6)-arene)RuCl(κ(2)-N,N-HL(1))]Cl differing in the arene moiety have been studied in depth in terms of thermodynamic dissociation constants, aquation kinetic constants, and DNA binding measurements. The biologically most active compound is the p-cym derivative, which strongly destabilizes the DNA double helix, whereas those with bz and phoxet have only a small effect on the stability of the DNA double helix. Moreover, the inhibitory activity of several compounds toward CDK1 has also been evaluated. The DNA binding ability of some of the studied compounds and their CDK1 inhibitory effect suggest a multitarget mechanism for their biological activity. Show less

New cationic, half-sandwich Ru(II) arene compounds of general formula [(η(6)-arene)RuCl(κ(2)-N,N-L)]X (where L are functionalized phenanthrolines such as 1,10-phenanthroline-5-amine (aphen); 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline (ephen); or 4,7-dihydroxy-1,10-phenanthroline (dhphen)) have been prepared to study their anticancer potential. All the isolated complexes have been fully characterized by spectroscopic and analytical techniques. The structure of endo-[(η(6)-p-cymene)RuCl(κ(2)-N,N-ephen)]BF4, [2a](BF4), has been determined by X-ray crystallography. The in vitro cytotoxicity of the aphen and ephen phenanthrolines and their Ru derivatives [(η(6)-p-cymene)RuCl(κ(2)-N,N-L)]Cl ([1a]Cl and [2a]Cl, respectively) assessed in tumour cell lines has shown that the free ligands are more active than the organometallic products, with aphen being the most potent specimen. Furthermore, the binding interaction of both [1a]Cl and aphen with calf thymus DNA (CT-DNA) has been investigated using a variety of thermodynamic and kinetic techniques. The aphen free ligand intercalates into DNA at low ligand content, whereas [1a]Cl forms with DNA a bifunctional partially intercalated-covalent complex, in which the intercalation constant is nearly three orders of magnitude lower than that of aphen. This finding demonstrates that the covalent binding noticeably weakens the intercalation, a feature presumably related to the higher cytotoxic activity of aphen relative to that of [1a]Cl. Show less

The synthesis and full characterization of the new aqua-complex [(η(6)-p-cymene)Ru(OH2)(κ(2)-N,N-2-pydaT)](BF4)2, [2](BF4)2, and the nucleobase derivative [(η(6)-p-cymene)Ru(9-MeG)(κ(2)-N,N-2-pydaT)](BF4)2, [4](PF6)2, where 2-pydaT = 2,4-diamino-6-(2-pyridyl)-1,3,5-triazine and 9-MeG = 9-methylguanine, are reported here. The crystal structures of both [4](PF6)2 and the chloro complex [(η(6)-p-cymene)RuCl(κ(2)-N,N-2-pydaT)](PF6), [1](PF6), have been elucidated by X-ray diffraction. The former provided relevant information regarding the interaction of the metallic fragment [(η(6)-p-cymene)Ru(κ(2)-N,N-2-pydaT)](2+) and a simple model of DNA. NMR and kinetic absorbance studies have proven that the aqua-complex [2](BF4)2 binds to the N7 site of guanine in nucleobases, nucleotides, or DNA. A stable bifunctional interaction (covalent and partially intercalated) between the [(η(6)-p-cymene)Ru(κ(2)-N,N-2-pydaT)](2+) fragment and CT-DNA has been corroborated by kinetic, circular dichroism, viscometry, and thermal denaturation experiments. The reaction mechanism entails the very fast formation of the Ru-O-(PO3) linkage prior to the fast intercalation of the 2-pydaT fragment. Then, a Ru-N7-(G) covalent bond is formed at the expense of the Ru-O-(PO3) bond, yielding a bifunctional complex. The dissociation rate of the intercalated fragment is slow, and this confers additional interest to [2](BF4)2 in view of the likely correlation between slow dissociation and biological activity, on the assumption that DNA is the only biotarget. Furthermore, [2](BF4)2 displays notable pH-dependent cytotoxic activity in human ovarian carcinoma cells (A2780, IC50 = 11.0 μM at pH = 7.4; IC50 = 6.58 μM at pH = 6.5). What is more, complex [2](BF4)2 is not cross-resistant with cisplatin, exhibiting a resistance factor, RF(A2780cis), of 0.28, and it shows moderate selectivity toward the cancer cell lines, in particular, A2780cis (IC50 = 3.0 5 ± 0.08 μM), relative to human lung fibroblast cells (MRC-5; IC50 = 24 μM), the model for healthy cells. Show less

Progress in an understanding of the genetic basis of cancer coupled to molecular pharmacology of potential new anticancer drugs calls for new approaches that are able to address key issues in the drug development process, including pharmacokinetic (PK) and pharmacodynamic (PD) relationships. The incorporation of predictive preclinical PK/PD models into rationally designed early-stage clinical trials offers a promising way to relieve a significant bottleneck in the drug discovery pipeline. The aim of the current review is to discuss some considerations for how quantitative PK and PD analyses for anticancer drugs may be conducted and integrated into a global translational effort, and the importance of examining drug disposition and dynamics in target tissues to support the development of preclinical PK/PD models that can be subsequently extrapolated to predict pharmacologic characteristics in patients. In this article, we describe three different physiologically based (PB) PK modeling approaches, i.e., the whole-body PBPK model, the hybrid PBPK model, and the two-pore model for macromolecules, as well as their applications. General conclusions are that greater effort should be made to generate more clinical data that could validate scaled preclinical PB-PK/PD tumor-based models and, thus, stimulate a framework for preclinical to clinical translation. Finally, given the innovative techniques to measure tissue drug concentrations and associated biomarkers of drug responses, development of predictive PK/PD models will become a standard approach for drug discovery and development. Show less