The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.

Synthesis, Characterization, and in Vitro Anticancer Activity of Copper and Zinc Bis(Thiosemicarbazone) Complexes.

A series of eight bis(thiosemicarbazone) ligands and 16 of their respective copper(II) and zinc(II) complexes containing a combination of hydrogen, methyl, pyridyl, phenyl, and/or ethyl substituents at the diimine position of the ligand backbone were synthesized and characterized. The objective of this study was to identify the structure-activity relationships within a series of analogues with different substituents at the diimine position of the backbone and at the terminal N atom. The Cu(II) complexes Cu(GTSM2), Cu(GTSCM), Cu(PyTSM2), Cu(EMTSM2) and Cu(PGTSM2) demonstrated a distorted square planar geometry, while the Zn(II) complexes Zn(ATSM2)(DMSO), Zn(PyTSM2)(DMSO), and Zn(PGTSM2)(H2O) formed a distorted square pyramidal geometry. Cyclic voltammetry showed that the Cu(II) complexes display quasi-reversible electrochemistry. Of the agents, Cu(II) glyoxal bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(GTSM2)] and Cu(II) diacetyl bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(ATSM2)] demonstrated the greatest antiproliferative activity against tumor cells. Substitutions at the diimine position and at the terminal N atom with hydrophobic moieties markedly decreased their antiproliferative activity. Complexation of the bis(thiosemicarbazones) with Zn(II) generally decreased their antiproliferative activity, suggesting the Zn(II) complex did not act as a chaperone to deliver the ligand intracellularly, in contrast to similar bis(thiosemicarbazone) cobalt(III) complexes [King et al. Inorg. Chem. 2017, 56, 6609-6623]. However, five of the eight bis(thiosemicarbazone) Cu(II) complexes maintained or increased their antiproliferative activity, relative to the ligand alone, and a mechanism of Cu-induced oxidative stress is suggested. Surprisingly, relative to normoxic growth conditions, hypoxia that is found in the tumor microenvironment decreased the antiproliferative efficacy of most bis(thiosemicarbazones) and their copper complexes. This was independent of the potential hypoxia-selectivity mediated by Cu(II/I) redox potentials. These results provide structure-activity relationships useful for the rational design of bis(thiosemicarbazone) anticancer agents.

Novel SPME fibers based on a plastic support for determination of plasma protein binding of thiosemicarbazone metal chelators: a case example of DpC, an anti-cancer drug that entered clinical trials.

Solid-phase microextraction (SPME) is an alternative method to dialysis and ultrafiltration for the determination of plasma protein binding (PPB) of drugs. It is particularly advantageous for complicated analytes where standard methods are not applicable. Di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) is a lead compound of novel thiosemicarbazone anti-cancer drugs, which entered clinical trials in 2016. However, this agent exhibited non-specific binding on filtration membranes and had intrinsic chelation activity, which precluded standard PPB methods. In this study, using a simple and fast procedure, we prepared novel SPME fibers for extraction of DpC based on a metal-free, silicon string support, covered with C18 sorbent. Reproducibility of the preparation process was demonstrated by the percent relative standard deviation (RSD) of ≤ 9.2% of the amount of DpC extracted from PBS by several independently prepared fibers. The SPME procedure was optimized by evaluating extraction and desorption time profiles. Suitability of the optimized protocol was verified by examining reproducibility, linearity, and recovery of DpC extracted from PBS or plasma. All samples extracted by SPME were analyzed using an optimized and validated UHPLC-MS/MS method. The developed procedure was applied to the in vitro determination of PPB of DpC at two clinically relevant concentrations (500 and 1000 ng/mL). These studies showed that DpC is highly bound to plasma proteins (PPB ≥ 88%) and this did not differ significantly between both concentrations tested. This investigation provides novel data in the applicability of SPME for the determination of PPB of chelators, as well as useful information for the clinical development of DpC. Graphical abstract.

Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and What Is Yet to Come.

Cancer is a leading cause of death in many countries around the world. However, the efficacy of current standard treatments for a variety of cancers is suboptimal. First, most cancer treatments lack specificity, meaning that these treatments affect both cancer cells and their normal counterparts. Second, many anticancer agents are highly toxic, and thus, limit their use in treatment. Third, a number of cytotoxic chemotherapeutics are highly hydrophobic, which limits their utility in cancer therapy. Finally, many chemotherapeutic agents exhibit short half-lives that curtail their efficacy. As a result of these deficiencies, many current treatments lead to side effects, noncompliance, and patient inconvenience due to difficulties in administration. However, the application of nanotechnology has led to the development of effective nanosized drug delivery systems known commonly as nanoparticles. Among these delivery systems, lipid-based nanoparticles, particularly liposomes, have shown to be quite effective at exhibiting the ability to: 1) improve the selectivity of cancer chemotherapeutic agents; 2) lower the cytotoxicity of anticancer drugs to normal tissues, and thus, reduce their toxic side effects; 3) increase the solubility of hydrophobic drugs; and 4) offer a prolonged and controlled release of agents. This review will discuss the current state of lipid-based nanoparticle research, including the development of liposomes for cancer therapy, different strategies for tumor targeting, liposomal formulation of various anticancer drugs that are commercially available, recent progress in liposome technology for the treatment of cancer, and the next generation of lipid-based nanoparticles.

Identification of differential phosphorylation and sub-cellular localization of the metastasis suppressor, NDRG1.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), exhibits pleiotropic activity, inhibiting metastasis of various tumor-types, while being correlated with metastasis in others. Notably, NDRG1 phosphorylation and cleavage are associated with its function, although it is unclear if these modifications occur universally, or selectively, in different cancer cell-types and if it contributes to its pleiotropy. Considering the suggested DNA repair role of nuclear NDRG1, the effects of the above post-translational modifications on its nuclear localization was examined. Herein, the full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus-directed antibody, while only the FL isoform was identified using an N-terminus-directed antibody. For the first time, we demonstrate that the expression of the NDRG1 FL and T forms occurs in all cancer cell-types examined, as does its phosphorylation (p-NDRG1) at Ser330 and Thr346. The FL isoform localized highly in the nucleus compared to the T isoform. Moreover, p-NDRG1 (Ser330) was also markedly localized in the nucleus, while p-NDRG1 (Thr346) was predominantly cytoplasmic in all cell-types. These results indicate the N-terminus region and phosphorylation at Ser330 could be crucial for NDRG1 nuclear localization and function. PTEN silencing indicated that p-NDRG1 (Thr346) could be regulated differentially in different tumor cell-types, indicating PTEN may be involved in the mechanism(s) underlying the pleiotropic activity of NDRG1. Finally, therapeutics of the di-2-pyridylketone thiosemicarbazone class increased nuclear NDRG1 isoforms (FL and T) detected by the C-terminus-directed antibody in HepG2 cells, while having no significant effect in PC3 cells, indicating differential activity depending on the cell-type.

Novel Thiosemicarbazones Inhibit Lysine-Rich Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1) Coisolated (LYRIC) and the LYRIC-Induced Epithelial-Mesenchymal Transition via Upregulation of N-Myc Downstream-Regulated Gene 1 (NDRG1).

Tumor necrosis factor α (TNFα) plays a vital role in cancer progression as it is associated with inflammation and promotion of cancer angiogenesis and metastasis. The effects of TNFα are mediated by its downstream target, the oncogene lysine-rich CEACAM1 coisolated protein (LYRIC, also known as metadherin or astrocyte elevated gene-1). LYRIC plays an important role in activating the nuclear factor-ĸB (NF-κB) signaling pathway, which controls multiple cellular processes, including proliferation, apoptosis, migration, etc. In contrast, the metastasis suppressor N-myc downstream regulated gene 1 (NDRG1) has the opposite effect on the NF-κB pathway, being able to inhibit NF-κB activation and reduce angiogenesis, proliferation, migration, and cancer cell invasion. These potent anticancer properties make NDRG1 an ideal therapeutic target. Indeed, a novel class of thiosemicarbazone anticancer agents that target this molecule has been developed; the lead agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, has recently entered clinical trials for advanced and resistant cancers. To further elucidate the interaction between NDRG1 and oncogenic signaling, this study for the first time assessed the effects of NDRG1 on the tumorigenic properties of TNFα and its downstream target, LYRIC. We have demonstrated that NDRG1 inhibits the TNFα-mediated epithelial-to-mesenchymal transition. Further, NDRG1 also potently inhibited LYRIC expression, with a negative feedback loop existing between these two molecules. Examining the mechanism involved, we demonstrated that NDRG1 inhibited phosphatidylinositol 3-kinase/AKT signaling, leading to reduced levels of the LYRIC transcriptional activator, c-Myc. Finally, we demonstrated that novel thiosemicarbazones that upregulate NDRG1 also inhibit LYRIC expression, further highlighting their marked potential for cancer treatment.

The Metastasis Suppressor, N-MYC Downstream-regulated Gene-1 (NDRG1), Down-regulates the ErbB Family of Receptors to Inhibit Downstream Oncogenic Signaling Pathways.

N-MYC downstream-regulated gene-1 (NDRG1) is a potent growth and metastasis suppressor that acts through its inhibitory effects on a wide variety of cellular signaling pathways, including the TGF-ß pathway, protein kinase B (AKT)/PI3K pathway, RAS, etc. To investigate the hypothesis that its multiple effects could be regulated by a common upstream effector, the role of NDRG1 on the epidermal growth factor receptor (EGFR) and other members of the ErbB family, namely human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3), was examined. We demonstrate that NDRG1 markedly decreased the expression and activation of EGFR, HER2, and HER3 in response to the epidermal growth factor (EGF) ligand, while also inhibiting formation of the EGFR/HER2 and HER2/HER3 heterodimers. In addition, NDRG1 also decreased activation of the downstream MAPKK in response to EGF. Moreover, novel anti-tumor agents of the di-2-pyridylketone class of thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, which markedly up-regulate NDRG1, were found to inhibit EGFR, HER2, and HER3 expression and phosphorylation in cancer cells. However, the mechanism involved appeared dependent on NDRG1 for di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, but was independent of this metastasis suppressor for di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone. This observation demonstrates that small structural changes in thiosemicarbazones result in marked alterations in molecular targeting. Collectively, these results reveal a mechanism for the extensive downstream effects on cellular signaling attributed to NDRG1. Furthermore, this study identifies a novel approach for the treatment of tumors resistant to traditional EGFR inhibitors.

Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).

The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-ß-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC(50)) by 140-fold. On the other hand, cholesterol extraction using methyl-ß-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics.

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.

Frataxin and the molecular mechanism of mitochondrial iron-loading in Friedreich's ataxia.

The mitochondrion is a major site for the metabolism of the transition metal, iron, which is necessary for metabolic processes critical for cell vitality. The enigmatic mitochondrial protein, frataxin, is known to play a significant role in both cellular and mitochondrial iron metabolism due to its iron-binding properties and its involvement in iron-sulfur cluster (ISC) and heme synthesis. The inherited neuro- and cardio-degenerative disease, Friedreich's ataxia (FA), is caused by the deficient expression of frataxin that leads to deleterious alterations in iron metabolism. These changes lead to the accumulation of inorganic iron aggregates in the mitochondrial matrix that are presumed to play a key role in the oxidative damage and subsequent degenerative features of this disease. Furthermore, the concurrent dys-regulation of cellular antioxidant defense, which coincides with frataxin deficiency, exacerbates oxidative stress. Hence, the pathogenesis of FA underscores the importance of the integrated homeostasis of cellular iron metabolism and the cytoplasmic and mitochondrial redox environments. This review focuses on describing the pathogenesis of the disease, the molecular mechanisms involved in mitochondrial iron-loading and the dys-regulation of cellular antioxidant defense due to frataxin deficiency. In turn, current and emerging therapeutic strategies are also discussed.

Letter to the Editor: "Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques".

n/a.

Novel thiosemicarbazones regulate the signal transducer and activator of transcription 3 (STAT3) pathway: inhibition of constitutive and interleukin 6-induced activation by iron depletion.

Pharmacologic manipulation of metal pools in tumor cells is a promising strategy for cancer treatment. Here, we reveal how the iron-binding ligands desferrioxamine (DFO), di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibit constitutive and interleukin 6-induced activation of signal transducer and activator of transcription 3 (STAT3) signaling, which promotes proliferation, survival, and metastasis of cancer cells. We demonstrate that DFO, Dp44mT, and DpC significantly decrease constitutive phosphorylation of the STAT3 transcription factor at Tyr705 in the pancreatic cancer cell lines PANC-1 and MIAPaCa-2 as well as the prostate cancer cell line DU145. These compounds also significantly decrease the dimerized STAT3 levels, the binding of nuclear STAT3 to its target DNA, and the expression of downstream targets of STAT3, including cyclin D1, c-myc, and Bcl-2. Examination of upstream mediators of STAT3 in response to these ligands has revealed that Dp44mT and DpC could significantly decrease activation of the nonreceptor tyrosine kinase Src and activation of cAbl in DU145 and MIAPaCa-2 cells. In contrast to the effects of Dp44mT, DpC, or DFO on inhibiting STAT3 activation, the negative control compound di-2-pyridylketone 2-methyl-3-thiosemicarbazone, or the DFO:Fe complex, which cannot bind cellular iron, had no effect. This demonstrates the role of iron-binding in the activity observed. Immunohistochemical staining of PANC-1 tumor xenografts showed a marked decrease in STAT3 in the tumors of mice treated with Dp44mT or DpC compared with the vehicle. Collectively, these studies demonstrate suppression of STAT3 activity by iron depletion in vitro and in vivo, and reveal insights into regulation of the critical oncogenic STAT3 pathway.

The metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), inhibits stress-induced autophagy in cancer cells.

N-myc downstream regulated gene 1 (NDRG1) is a potent metastasis suppressor with an undefined role in the stress response. Autophagy is a pro-survival pathway and can be regulated via the protein kinase-like endoplasmic reticulum kinase (PERK)/eIF2α-mediated endoplasmic reticulum (ER) stress pathway. Hence, we investigated the role of NDRG1 in stress-induced autophagy as a mechanism of inhibiting metastasis via the induction of apoptosis. As thiosemicarbazone chelators induce stress and up-regulate NDRG1 to inhibit metastasis, we studied their effects on the ER stress response and autophagy. This was important to assess, as little is understood regarding the role of the stress induced by iron depletion and its role in autophagy. We observed that the chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which forms redox-active iron and copper complexes, effectively induced ER stress as shown by activation of the PERK/eIF2α pathway. Dp44mT also increased the expression of the autophagic marker, LC3-II, and this was dependent on activation of the PERK/eIF2α axis, as silencing PERK prevented LC3-II accumulation. The effect of Dp44mT on LC3-II expression was at least partially due to iron-depletion, as this effect was also demonstrated with the classical iron chelator, desferrioxamine (DFO), and was not observed for the DFO-iron complex. NDRG1 overexpression also inhibited basal autophagic initiation and the ER stress-mediated autophagic pathway via suppression of the PERK/eIF2α axis. Moreover, NDRG1-mediated suppression of the pro-survival autophagic pathway probably plays a role in its anti-metastatic effects by inducing apoptosis. In fact, multiple pro-apoptotic markers were increased, whereas anti-apoptotic Bcl-2 was decreased upon NDRG1 overexpression. This study demonstrates the role of NDRG1 as an autophagic inhibitor that is important for understanding its mechanism of action.

Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy.

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.

Expanding horizons in iron chelation and the treatment of cancer: role of iron in the regulation of ER stress and the epithelial-mesenchymal transition.

Cancer is a major public health issue and, despite recent advances, effective clinical management remains elusive due to intra-tumoural heterogeneity and therapeutic resistance. Iron is a trace element integral to a multitude of metabolic processes, including DNA synthesis and energy transduction. Due to their generally heightened proliferative potential, cancer cells have a greater metabolic demand for iron than normal cells. As such, iron metabolism represents an important "Achilles' heel" for cancer that can be targeted by ligands that bind and sequester intracellular iron. Indeed, novel thiosemicarbazone chelators that act by a "double punch" mechanism to both bind intracellular iron and promote redox cycling reactions demonstrate marked potency and selectivity in vitro and in vivo against a range of tumours. The general mechanisms by which iron chelators selectively target tumour cells through the sequestration of intracellular iron fall into the following categories: (1) inhibition of cellular iron uptake/promotion of iron mobilisation; (2) inhibition of ribonucleotide reductase, the rate-limiting, iron-containing enzyme for DNA synthesis; (3) induction of cell cycle arrest; (4) promotion of localised and cytotoxic reactive oxygen species production by copper and iron complexes of thiosemicarbazones (e.g., Triapine(®) and Dp44mT); and (5) induction of metastasis and tumour suppressors (e.g., NDRG1 and p53, respectively). Emerging evidence indicates that chelators can further undermine the cancer phenotype via inhibiting the epithelial-mesenchymal transition that is critical for metastasis and by modulating ER stress. This review explores the "expanding horizons" for iron chelators in selectively targeting cancer cells.

The renaissance of polypharmacology in the development of anti-cancer therapeutics: Inhibition of the "Triad of Death" in cancer by Di-2-pyridylketone thiosemicarbazones.

Cancer is a disease that is a "moving target", since as the condition progresses, the molecular targets change and evolve. Moreover, due to clonal selection, a specific anti-cancer drug with one molecular target may only be effective for a limited time period before drug resistance results and the agent becomes ineffective. Hence, the concept of an anti-tumor therapeutic exhibiting polypharmacology can be highly advantageous, rather than a therapeutic obstacle. A novel class of agents possessing these desirable properties are the di-2-pyridylketone thiosemicarbazones, which bind iron and copper to affect a variety of critical molecular targets in tumors. In fact, these compounds possess multiple properties that enable them to overcome the "triad of death" in cancer, namely: primary tumor growth, drug resistance and metastasis. In fact, at the molecular level, their potent anti-oncogenic activity includes: up-regulation of the metastasis suppressor, N-myc downstream regulated gene 1; up-regulation of the tumor suppressor, PTEN; down-regulation of the proto-oncogene, cyclin D1; inhibition of the rate-limiting step in DNA synthesis catalyzed by ribonucleotide reductase; and the inhibition of multiple oncogenic signaling pathways, e.g., Ras/MAPK signaling, protein kinase B (AKT)/phosphatidylinositol-3-kinase, ROCK/pMLC2, etc. This Perspective article discusses the advantages of incorporating polypharmacology into anti-cancer drug design using the di-2-pyridylketone thiosemicarbazones as a pertinent example.

Potent antimycobacterial activity of the pyridoxal isonicotinoyl hydrazone analog 2-pyridylcarboxaldehyde isonicotinoyl hydrazone: a lipophilic transport vehicle for isonicotinic acid hydrazide.

The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

Effect of the piperazine unit and metal-binding site position on the solubility and anti-proliferative activity of ruthenium(II)- and osmium(II)- arene complexes of isomeric indolo[3,2-c]quinoline-piperazine hybrids.

In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline-piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline-piperazine hybrids L(1-3) were prepared in situ and isolated as six ruthenium and osmium complexes [(η(6)-p-cymene)M(L(1-3))Cl]Cl, where L(1) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L(2) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L(3) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV-vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl-[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure-activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline-piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents.

Simultaneous determination of the novel thiosemicarbazone anti-cancer agent, Bp4eT, and its main phase I metabolites in plasma: application to a pilot pharmacokinetic study in rats.

Novel thiosemicarbazone metal chelators are extensively studied anti-cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC-MS/MS method for the simultaneous quantification of 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1-E and M1-Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C18 column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid-phase extraction on 96-well plates. This method was validated over the concentration range of 0.18-2.80 µM for Bp4eT, 0.02-0.37 µM for both M1-E and M1-Z, and 0.10-1.60 µM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration-time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti-cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class.

Cellular uptake of the antitumor agent Dp44mT occurs via a carrier/receptor-mediated mechanism.

The chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) shows potent and selective anticancer and antimetastatic activity. However, the mechanism by which it is initially transported into cells to induce cytotoxicity is unknown. Hence, the current investigation examined the cellular uptake of ¹4C-Dp44mT relative to two structurally related ligands, namely the aroylhydrazone ¹4C-pyridoxal isonicotinoyl hydrazone (¹4C-PIH) and the thiosemicarbazone (¹4C-2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (¹4C-Bp4eT). In marked contrast to the cellular uptake of ¹4C-PIH and ¹4C-Bp4eT, which were linear as a function of concentration, ¹4C-Dp44mT uptake was saturable using SK-N-MC neuroepithelioma cells (Bmax, 4.28 × 107 molecules of chelator/cell; and Kd, 2.45 µM). Together with the fact that ¹4C-Dp44mT uptake was temperature-dependent and significantly (P < 0.01) decreased by competing unlabeled Dp44mT, these observations indicated a saturable transport mechanism consistent with carrier/receptor-mediated transport. Other unlabeled ligands that shared the saturated N4 structural moiety with Dp44mT significantly (P < 0.01) inhibited ¹4C-Dp44mT uptake, illustrating its importance for carrier/receptor recognition. Nevertheless, unlabeled Dp44mT most markedly decreased (¹4C-Dp44mT uptake, demonstrating that the putative carrier/receptor shows high selectivity for Dp44mT. Interestingly, in contrast to ¹4C-Dp44mT, uptake of its Fe complex [Fe(¹4C-Dp44mT)2] was not saturable as a function of concentration and was much greater than the ligand alone, indicating an alternate mode of transport. Studies examining the tissue distribution of ¹4C-Dp44mT injected intravenously into a mouse tumor model demonstrated the ¹4C label was primarily identified in the excretory system. Collectively, these findings examining the mechanism of Dp44mT uptake and its distribution and excretion have clinical implications for its bioavailability and uptake in vivo.