An Undefined Interaction between Polyamines and Heat Shock Proteins Leads to Cellular Protection in Plasmodium falciparum and Proliferating Cells in Various Organisms.

Environmental stimuli can distress the internal reaction of cells and their normal function. To react promptly to sudden environmental changes, a cascade of heat shock proteins (Hsps) functions to protect and act as housekeepers inside the cells. In parallel to the heat shock response, the metabolic polyamine (PA) status changes. Here, we discuss possible ways of putative interactions between Hsps and polyamines in a wide lineage of eukaryotic model organisms with a particular focus on parasitic protozoa such as Plasmodium falciparum (P. falciparum). The supposed interaction between polyamines and Hsps may protect the parasite from the sudden change in temperature during transmission from the female Anopheles mosquito to a human host. Recent experiments performed with the spermidine mimetic inhibitor 15-deoxyspergualine in Plasmodium in vitro cultures show that the drug binds to the C-terminal EEVD motif of Hsp70. This leads to inhibition of protein biosynthesis caused by prevention of eIF5A2 phosphorylation and eukaryotic initiation factor 5A (eIF5A) modification. These observations provide further evidence that PAs are involved in the regulation of protein biosynthesis of Hsps to achieve a protective effect for the parasite during transmission.

Hypusinated EIF5A as a feasible drug target for Advanced Medicinal Therapies in the treatment of pathogenic parasites and therapy-resistant tumors.

Cancer drug resistance, in particular in advanced stages such as metastasis and invasion is an emerging problem. Moreover, drug resistance of parasites causing poverty-related diseases is an enormous, global challenge for drug development in the future. To circumvent this problem of increasing resistance, the development of either novel small compounds or Advanced Medicinal Therapies have to be fostered. Polyamines have many fundamental cellular functions like DNA stabilization, protein translation, ion channel regulation, autophagy, apoptosis and mostly important, cell proliferation. Consequently, many antiproliferative drugs can be commonly administered either in cancer therapy or for the treatment of pathogenic parasites. Most important for cell proliferation is the triamine spermidine, since it is an important substrate in the biosynthesis of the posttranslational modification hypusine in eukaryotic initiation factor 5A (EIF5A). To date, no small compound has been identified that directly inhibits the precursor protein EIF5A. Moreover, only a few small molecule inhibitors of the two biosynthetic enzymes, i.e. deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) have been functionally characterized. However, it is evident that only some of the compounds have been applied in translational approaches, i.e. in murine models to analyze the function of this modified protein in cell proliferation. In recent years, the pharmaceutical industry shifted from small molecules beyond traditional pharmacology to new tools and methods to treat disorders involving signaling deregulation. In this review, we evaluate translational approaches on inhibition of EIF5A hypusination in pathogenic parasites and therapy-resistant tumors and discuss its feasibility for an application in Advanced Medicinal Therapies.

Bovine Serum Amine Oxidase and Polyamine Analogues: Chemical Synthesis and Biological Evaluation Integrated with Molecular Docking and 3-D QSAR Studies.

Natural polyamines (PAs) are key players in cellular homeostasis by regulating cell growth and proliferation. Several observations highlight that PAs are also implicated in pathways regulating cell death. Indeed, the PA accumulation cytotoxic effect, maximized with the use of bovine serum amine oxidase (BSAO) enzyme, represents a valuable strategy against tumor progression. In the present study, along with the design, synthesis, and biological evaluation of a series of new spermine (Spm) analogues (1-23), a mixed structure-based (SB) and ligand-based (LB) protocol was applied. Binding modes of BSAO-PA modeled complexes led to clarify electrostatic and steric features likely affecting the BSAO-PA biochemical kinetics. LB and SB three-dimensional quantitative structure-activity relationship (Py-CoMFA and Py-ComBinE) models were developed by means of the 3d-qsar.com portal, and their analysis represents a strong basis for future design and synthesis of PA BSAO substrates for potential application in oxidative stress-induced chemotherapy.

Acidic Shift of Optimum pH of Bovine Serum Amine Oxidase upon Immobilization onto Nanostructured Ferric Tannates.

Protein-nanoparticle hybrids represent entities characterized by emerging biological properties that can significantly differ from those of the parent components. Herein, bovine serum amine oxidase (i.e., BSAO) was immobilized onto a magnetic nanomaterial constituted of surface active maghemite nanoparticles (i.e., SAMNs, the core), surface-modified with tannic acid (i.e., TA, the shell), to produce a biologically active ternary hybrid (i.e., SAMN@TA@BSAO). In comparison with the native enzyme, the secondary structure of the immobilized BSAO responded to pH variations sensitively, resulting in a shift of its optimum activity from pH 7.2 to 5.0. Conversely, the native enzyme structure was not influenced by pH and its activity was affected at pH 5.0, i.e., in correspondence with the best performances of SAMN@TA@BSAO. Thus, an extensive NMR study was dedicated to the structure-function relationship of native BSAO, confirming that its low activity below pH 6.0 was ascribable to minimal structural modifications not detected by circular dichroism. The generation of cytotoxic products, such as aldehydes and H2O2, by the catalytic activity of SAMN@TA@BSAO on polyamine oxidation is envisaged as smart nanotherapy for tumor cells. The present study supports protein-nanoparticle conjugation as a key for the modulation of biological functions.

Anticancer Cytotoxic Activity of Bispidine Derivatives Associated with the Increasing Catabolism of Polyamines.

Polyamine (PA) catabolism is often reduced in cancer cells. The activation of this metabolic pathway produces cytotoxic substances that might cause apoptosis in cancer cells. Chemical compounds able to restore the level of PA catabolism in tumors could become potential antineoplastic agents. The search for activators of PA catabolism among bicyclononan-9-ones is a promising strategy for drug development. The aim of the study was to evaluate the biological activity of new 3,7-diazabicyclo[3.3.1]nonan-9-one derivatives that have antiproliferative properties by accelerating PA catabolism. Eight bispidine derivatives were synthetized and demonstrated the ability to activate PA catabolism in regenerating rat liver homogenates. However, only three of them demonstrated a potent ability to decrease the viability of cancer cells in the MTT assay. Compounds 4c and 4e could induce apoptosis more effectively in cancer HepG2 cells rather than in normal WI-38 fibroblasts. The lead compound 4e could significantly enhance cancer cell death, but not the death of normal cells if PAs were added to the cell culture media. Thus, the bispidine derivative 4e 3-(3-methoxypropyl)-7-[3-(1H-piperazin-1-yl)ethyl]-3,7-diazabicyclo[3.3.1]nonane could become a potential anticancer drug substance whose mechanism relies on the induction of PA catabolism in cancer cells.

Involvement of the biogenic active amine agmatine in mitochondrial membrane permeabilization and release of pro-apoptotic factors.

Agmatine (AGM) produces a dual effect on the mitochondrial permeability transition (MPT) mechanism in rat liver mitochondria: at low concentrations, it induces the phenomenon, at high ones, inhibits it. The prevention at high concentrations is evidenced by the significant inhibition of mitochondrial swelling induced by Ca2+ and phosphate; in this condition, AGM both prevents the release of Apoptosis Inducing Factor (AIF) and enhances the release of other pro-apoptotic factors, such as cytochrome c (cyt c) and Smac/DIABLO. As these factors are released without MPT induction, the involvement of mitochondrial outer membrane permeabilization (MOMP) could be hypothesized. Cyclosporin A (CsA), a powerful inhibitor of MPT, enhanced the AGM-mediated inhibition of swelling, and surprisingly, prevented the release of cyt c and Smac/DIABLO. In the presence of Ca2+, AGM also activated the Bcl-2 family protein Bax, a key factor in inducing MOMP, which is inactivated by CsA. Together with the voltage-dependent anion channel (VDAC), Bax forms channels in the outer membrane further supporting the involvement of MOMP in the release of pro-apoptotic factors. In view of the fact that VDAC was inactivated by ruthenium red, which in turn inhibited the release of cyt c, it can be hypothesized that, on the one hand, AGM inhibits MPT induction and, on the other, it selectively permeabilizes the outer membrane via MOMP induction.

The investigation of structure-activity relationship of polyamine-targeted synthetic compounds from different chemical groups.

The polyamine (PA) metabolism is involved in cell proliferation and differentiation. Increased cellular PA levels are observed in different types of cancers. Products of PA oxidation induce apoptosis in cancer cells. These observations open a perspective to exploit the enzymes of PA catabolism as a target for anticancer drug design. The substances capable to enhance PA oxidation may become potential anticancer agents. The goal of our study was to explore how the mode of ligand binding with a PA catabolic enzyme is associated with its stimulatory or inhibitory effect upon PA oxidation. Murine N1-acetylpolyamine oxidase (5LFO) crystalline structure was used for molecular docking with ligands of various chemical structures. In vitro experiments were carried out to evaluate the action of the tested compounds upon PA oxidative deamination in a cell-free test system from rat liver. Two amino acid residues (Aps211 and Tyr204) in the structure of 5LFO were found to be significant for binding with the tested compounds. 19 out of 51 screened compounds were activators and 17 were inhibitors of oxidative deamination of PA. Taken together, these results enabled to construct a recognition model with characteristic descriptors depicting activators and inhibitors. The general tendency indicated that a strong interaction with Asp211 or Tyr204 was rather typical for activators. The understanding of how the structure determines the binding mode of compounds with PA catabolic enzyme may help in explanation of their structure-activity relationship and thus promote structure-based drug design.

A novel comprehensive paradigm for the etiopathogenesis of multiple sclerosis: therapeutic approaches and future perspectives on its treatment.

It is well recognized that variation in the geographical distribution of prevalence of multiple sclerosis (MS) exists: increasing the latitude its prevalence increases as well, but the underlying causes of such dissimilarity still remained elusive as of today. Currently, the most accredited hypothesis is that the closer to the equator the more pronounced is the amount of sunlight which, in turn, increases the production of vitamin D. Cholecalciferol is indeed deficient in MS patients, but this factor does not explain by itself the etiopathogenesis of the disease. In the present study, to search for a pattern and provide a model of the disease's etiology consistent with this regional factor, as well with its changing ethnic, sex-ratio, lifestyle variations and the other unexplained aspects of MS, an extensive analysis of peer-reviewed literature and data was conducted. The arisen hypothesis was that, increasing the latitude, the factor that varies and can have the stronger effect on the human organism, is the continuous and ever-increasing diversity of the natural light-dark cycle. The consequent effort of the suprachiasmatic nucleus to entrain the organism's circadian rhythm affects the hypothalamic-pituitary-adrenal axis resulting in desynchronizing the central and peripheral circadian clocks and pathologizing the immunitary system. To verify such hypothesis, a theoretical framework of the etiopathogenesis, coherent with the gathered literature, was conceived and a demonstration to corroborate it was eventually devised and performed. The results underscored that people living in countries subjected to a further circadian disruptive factor, as daylight saving time, have a 6.35 times higher prevalence of MS than States placed on their same latitude that do not observe it, thus strongly supporting the hypothesis. As further reinforcement of the conclusions, it is worth mentioning that the levels of polyamines rise abruptly in autoimmune diseases. Moreover, among their numerous roles, these polycations participate to the regulation of the circadian clock so their sudden variation might disrupt it. Following these interesting findings, new perspectives in therapies are, therefore, proposed.

Enzyme self-assembly on naked iron oxide nanoparticles for aminoaldehyde biosensing.

The preservation of enzymatic activity is a fundamental requirement for exploiting hybrid nano-bio-conjugates, and the control over protein-nanoparticle interactions, leading to stable and catalytically active hybrids, represents the key for designing new biosensing platforms. In this scenario, surface active maghemite nanoparticles (SAMNs) represent a new class of naked magnetic nanoparticles, displaying peculiar electrocatalytic features and the ability to selectively bind proteins. Recombinant aminoaldehyde dehydrogenase from tomato (SlAMADH1) was used as a model protein, and successfully immobilized by self-assembly on the surface of naked SAMNs, where its enzymatic activity resulted preserved for more than 6 months. The hybrid nanomaterial (SAMN@SlAMADH1) was characterized by UV-Vis spectroscopy, mass spectrometry, and TEM microscopy, and applied for the development of a biosensor for the determination of aminoaldehydes in alcoholic beverages. Measurements were carried out in a low volume electrochemical flow cell comprising a SAMN modified carbon paste electrode for the coulometric determination of the NADH produced during the enzymatic catalysis. The present findings, besides representing the first example of an electrochemical biosensor for aminoaldehydes in an alcoholic matrix, open the door to the use of immobilized enzymes on naked metal oxides nanomaterials for biosensing.

Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase.

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1-9) or methoctramine (10-14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 µM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

Endogenous and food-derived polyamines: determination by electrochemical sensing.

Polyamines (PAs) are involved in a variety of fundamental physio-pathologic processes. The concentration of these polycations in organs and tissues depends on their endogenous production and oxidation rates, and on their intake from foods. Besides being largely accepted as markers for the progress of several pathologies, PAs may exert themselves different effects on humans, ranging from being positive to be drastically detrimental depending on the organism conditions. Thus, if the determination of polyamines content in tissue samples is of great importance as they could be indicators of several diseases, their quantification in food is fundamental for modulating the diet to respond to a specific human health status. Thus, the determination of PA content in food is increasingly urgent. Standard analytical methods for polyamine quantification are mainly based on chromatography, where high-performance liquid chromatography and gas chromatography are the most often used, involving pre-column or post-column derivatization techniques. Driven by the growing need for rapid in situ analyses, electrochemical biosensors, comprising various combinations of different enzymes or nanomaterials for the selective bio-recognition and detection, are emerging as competitors of standard detection systems. The present review is aimed at providing an up-to-date overview on the recent progresses in the development of sensors and biosensors for the detection of polyamines in human tissues and food samples. Basic principles of different electrochemical (bio)sensor formats are reported and the applications in human tissues and in foods was evidenced.

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

Ternary Hybrid γ-Fe2 O3 /Cr(VI) /Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue.

Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts.

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A.

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria which distinguish them from other amines. Enzymatic oxidative deamination of spermine by amine oxidases in tumor cells may produce reactive oxygen species, leading to transition pore opening and apoptosis. This process could be exploited as a new therapeutic strategy to combat cancer.

Spermine cycling in mitochondria is mediated by adenine nucleotide translocase activity: mechanism and pathophysiological implications.

Spermine, besides to be transported in mitochondria by an energy dependent electrophoretic mechanism, can be also released by two different mechanisms. The first one is induced in deenergizing conditions by FCCP or antimycin A and it is mediated by an electroneutral exchange spermine protons. The second one takes place in energizing conditions during the activity of the adenine nucleotide translocase and is mediated by an electroneutral symport mechanism involving the efflux in co-transport of spermine and phosphate and the exchange of exogenous ADP with endogenous ATP. The triggering of this mechanism permits an alternating cycling of spermine across the mitochondrial membrane, that is spermine is transported or released by energized mitochondria in the absence or presence of ATP synthesis, respectively. The physiological implications of this cycling of spermine are related to the induction or prevention of mitochondrial permeability transition and, consequently, on apoptosis or its prevention.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

New trans dichloro (triphenylphosphine)platinum(II) complexes containing N-(butyl),N-(arylmethyl)amino ligands: Synthesis, cytotoxicity and mechanism of action.

Some new platinum(II) complexes have been prepared, of general formula trans-[PtCl2(PPh3){NH(Bu)CH2Ar}], where the dimension of the Ar residue in the secondary amines has been varied from small phenyl to large pyrenyl group. The obtained complexes, tested in vitro towards a panel of human tumor cell lines showed an interesting antiproliferative effect on both cisplatin-sensitive and -resistant cells. For the most cytotoxic derivative 2a the investigation on the mechanism of action highlighted the ability to induce apoptosis on resistant cells and interestingly, to inhibit the catalytic activity of topoisomerase II.

Pathophysiological implications of mitochondrial oxidative stress mediated by mitochondriotropic agents and polyamines: the role of tyrosine phosphorylation.

Mitochondria, once merely considered as the "powerhouse" of cells, as they generate more than 90 % of cellular ATP, are now known to play a central role in many metabolic processes, including oxidative stress and apoptosis. More than 40 known human diseases are the result of excessive production of reactive oxygen species (ROS), bioenergetic collapse and dysregulated apoptosis. Mitochondria are the main source of ROS in cells, due to the activity of the respiratory chain. In normal physiological conditions, ROS generation is limited by the anti-oxidant enzymatic systems in mitochondria. However, disregulation of the activity of these enzymes or interaction of respiratory complexes with mitochondriotropic agents may lead to a rise in ROS concentrations, resulting in oxidative stress, mitochondrial permeability transition (MPT) induction and triggering of the apoptotic pathway. ROS concentration is also increased by the activity of amine oxidases located inside and outside mitochondria, with oxidation of biogenic amines and polyamines. However, it should also be recalled that, depending on its concentration, the polyamine spermine can also protect against stress caused by ROS scavenging. In higher organisms, cell signaling pathways are the main regulators in energy production, since they act at the level of mitochondrial oxidative phosphorylation and participate in the induction of the MPT. Thus, respiratory complexes, ATP synthase and transition pore components are the targets of tyrosine kinases and phosphatases. Increased ROS may also regulate the tyrosine phosphorylation of target proteins by activating Src kinases or phosphatases, preventing or inducing a number of pathological states.