From Monoamine Oxidase Inhibition to Antiproliferative Activity: New Biological Perspectives for Polyamine Analogs.

Monoamine oxidases (MAOs) are well-known pharmacological targets in neurological and neurodegenerative diseases. However, recent studies have revealed a new role for MAOs in certain types of cancer such as glioblastoma and prostate cancer, in which they have been found overexpressed. This finding is opening new frontiers for MAO inhibitors as potential antiproliferative agents. In light of our previous studies demonstrating how a polyamine scaffold can act as MAO inhibitor, our aim was to search for novel analogs with greater inhibitory potency for human MAOs and possibly with antiproliferative activity. A small in-house library of polyamine analogs (2-7) was selected to investigate the effect of constrained linkers between the inner amine functions of a polyamine backbone on the inhibitory potency. Compounds 4 and 5, characterized by a dianiline (4) or dianilide (5) moiety, emerged as the most potent, reversible, and mainly competitive MAO inhibitors (Ki < 1 µM). Additionally, they exhibited a high antiproliferative activity in the LN-229 human glioblastoma cell line (GI50 < 1 µM). The scaffold of compound 5 could represent a potential starting point for future development of anticancer agents endowed with MAO inhibitory activity.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody.

BACKGROUND: G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. METHODS: SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. RESULTS: The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. CONCLUSIONS: Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. GENERAL SIGNIFICANCE: The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Cardiolipin drives the catalytic activity of GPX4 on membranes: Insights from the R152H mutant.

The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to "positive feedback" in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.

Role of carboxylic group pattern on protein surface in the recognition of iron oxide nanoparticles: A key for protein corona formation.

The knowledge of protein-nanoparticle interplay is of crucial importance to predict the fate of nanomaterials in biological environments. Indeed, protein corona on nanomaterials is responsible for the physiological response of the organism, influencing cell processes, from transport to accumulation and toxicity. Herein, a comparison using four different proteins reveals the existence of patterned regions of carboxylic groups acting as recognition sites for naked iron oxide nanoparticles. Readily interacting proteins display a distinctive surface distribution of carboxylic groups, recalling the geometric shape of an ellipse. This is morphologically complementary to nanoparticles curvature and compatible with the topography of exposed FeIII sites laying on the nanomaterial surface. The recognition site, absent in non-interacting proteins, promotes the nanoparticle harboring and allows the formation of functional protein coronas. The present work envisages the possibility of predicting the composition and the biological properties of protein corona on metal oxide nanoparticles.

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3ε.

Ras-selective lethal small molecule 3 (RSL3), a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating the glutathione peroxidase glutathione peroxidase 4 (GPx4). Here, we report the purification of the protein indispensable for GPx4 inactivation by RSL3. Mass spectrometric analysis identified 14-3-3 isoforms as candidates, and recombinant human 14-3-3ε confirms the identification. The function of 14-3-3ε is redox-regulated. Moreover, overexpression or silencing of the gene coding for 14-3-3ε consistently controls the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein operating in cell signaling further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives.

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1.

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides.

Fast and simple method for the simultaneous evaluation of the capacity and efficiency of food antioxidants in trapping peroxyl radicals in an intestinal model system.

A simple oxygraphic method, for which the theoretical and experimental bases have been recently revised, has been successfully applied to evaluate the peroxyl radical chain-breaking characteristics of some typical food antioxidants in micelle systems, among which is a system that reproduces conditions present in the upper part of the digestive tract, where the absorption and digestion of lipids occur. This method permits one to obtain from a single experimental run the peroxyl radical trapping capacity (PRTC, that is, the number of moles of peroxyl radicals trapped by a given amount of food), the peroxyl radical trapping efficiency (PRTE, that is, the reciprocal of the amount of food that reduces to half the steady-state concentration of peroxyl radicals), and the half-life of the antioxidant ( t(1/2)) when only a small fraction of peroxyl radicals reacts with the antioxidants present in foods. Examples of application of the method to various types of foodstuffs have been reported, assessing the general validity of the method in the simple and fast evaluation of the above-reported fundamental antioxidant characteristics of foods.

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates.

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37 degrees C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is -OH>-OCH(3)>-H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.

Glutathione peroxidase 4-catalyzed reduction of lipid hydroperoxides in membranes: The polar head of membrane phospholipids binds the enzyme and addresses the fatty acid hydroperoxide group toward the redox center.

GPx4 is a monomeric glutathione peroxidase, unique in reducing the hydroperoxide group (-OOH) of fatty acids esterified in membrane phospholipids. This reaction inhibits lipid peroxidation and accounts for enzyme's vital role. Here we investigated the interaction of GPx4 with membrane phospholipids. A cationic surface near the GPx4 catalytic center interacts with phospholipid polar heads. Accordingly, SPR analysis indicates cardiolipin as the phospholipid with maximal affinity to GPx4. Consistent with the electrostatic nature of the interaction, KCl increases the KD. Molecular dynamic (MD) simulation shows that a -OOH posed in the core of the membrane as 13 - or 9 -OOH of tetra-linoleoyl cardiolipin or 15 -OOH stearoyl-arachidonoyl-phosphaphatidylcholine moves to the lipid-water interface. Thereby, the -OOH groups are addressed toward the GPx4 redox center. In this pose, however, the catalytic site facing the membrane would be inaccessible to GSH, but the consecutive redox processes facilitate access of GSH, which further primes undocking of the enzyme, because GSH competes for the binding residues implicated in docking. During the final phase of the catalytic cycle, while GSSG is produced, GPx4 is disconnected from the membrane. The observation that GSH depletion in cells induces GPx4 translocation to the membrane, is in agreement with this concept.

Red chicories as potent scavengers of highly reactive radicals: a study on their phenolic composition and peroxyl radical trapping capacity and efficiency.

Eight varieties of Cichorium genus vegetables (five heavily red colored, one red spotted, and two fully green) were investigated for their phenolic content (by HPLC and UV-vis spectrophotometry) and for their antioxidant activity. In particular, the capacity (that is, the amount of trapped peroxyl radicals) and the efficiency (that is, the amount of antioxidant necessary to halve the steady-state concentration of peroxyl radicals) were measured. All of the studied chicories are characterized by the presence of a large amount of hydroxybenzoic and hydroxycinnamic acids, whereas the red color is due to cyanidin glycosides. The presence of these phenolics in red chicories confers to them an exceptionally high peroxyl radical scavenging activity in terms of both capacity and efficiency, particularly in their early stage of growth, and makes this popular and low-cost foods comparable or superior to many foods having well-known antioxidant properties such as red wine, blueberry, and tomato.

Major phytochemicals in apple cultivars: contribution to peroxyl radical trapping efficiency.

Forty-one samples of apples (peel plus pulp), obtained from eight cultivars, were examined for concentration of some important phytochemicals and for antioxidant activity expressed as peroxyl radical trapping efficiency. Five major polyphenolic groups plus ascorbate were identified and quantified by HPLC in the apple varieties. Oligomeric and polymeric proanthocyanidins were found to be about two-thirds of total polyphenols. The antioxidant efficiency of the apple extracts and of representative pure compounds for each group of phytochemicals was measured in a micellar system mimicking lipid peroxidation in human plasma. Although the amount of polyphenols measured by HPLC is similar to that measured by standard methods, the antioxidant efficiency calculated on the basis of the contribution of the pure compounds was lower than the antioxidant efficiency of the apple extracts. The higher efficiency of apples appears to be strictly related to the overwhelming presence of oligomeric proanthocyanidins.

Interaction of copper with cysteine: stability of cuprous complexes and catalytic role of cupric ions in anaerobic thiol oxidation.

Copper complexes with cysteine have been investigated by optical spectroscopy, NMR and ESR. Cuprous ions strongly bind to the thiol group of Cys forming polymeric species with bridging thiolate sulfur according to a stoichiometry of about 1:1.2 and stability constant of the order of 10(10) M(-1). Cupric ions in the presence of cysteine, up to a ratio 0.45:1, are reduced to Cu(I) with stoichiometric production of cystine. The Cu(I) produced by this reaction is complexed by the excess of Cys. Trace amounts of Cu(II) exceeding the ratio 0.45:1 induce fast and complete oxidation of the Cys-Cu(I) complex to cystine with concomitant production of Cu(0) which precipitates. The experimental data are consistent with a mechanism by which Cu(II) oxidizes the complex Cys-Cu(I) to cystine producing aqueous Cu(I) which undergoes dismutation regenerating Cu(II). According to this mechanism the uncomplexed Cu(II) plays a catalytic oxidative role in the absence of molecular oxygen. The biological significance of these reactions is discussed.

Stable free radicals and peroxyl radical trapping capacity in red wines.

Thirty-two experimental red wines, obtained from eight cultivars and aged in bottles for 2 and 7 years, were examined for the presence of stable free radicals (SFR), for the peroxyl radical trapping capacity (PRTC), and for the concentrations of some important polyphenol families. Aging significantly increases SFR, polyphenol polymers with n > or = 5 (HMWP), and PRTC and is accompanied by a strong decrease of free anthocyanins. Multivariate regression analyses show that HMWP and SFR are independently associated with PRTC while HMWP and anthocyanins are independently associated with the formation of SFR. These results indicate that polymeric polyphenols generated from anthocyanins and proanthocyanidins during wine aging are able to convert highly reactive free radicals into nonreactive radicals through electron delocalization. The strict correlation between SFR and antioxidant activity that we found suggests that these characteristics are related to the functional properties of food.

Reaction rates of α-tocopheroxyl radicals confined in micelles and in human plasma lipoproteins.

α-Tocopherol, the main component of vitamin E, traps highly reactive radicals which otherwise might react with lipids present in plasmatic lipoproteins or in cell membranes. The α-tocopheroxyl radicals generated by this process have also a pro-oxidant action which is contrasted by their reaction with ascorbate or by bimolecular self-reaction (dismutation). The kinetics of this bimolecular self-reaction were explored in solution such as ethanol, and in heterogeneous systems such as deoxycholic acid micelles and in human plasma. According to ESR measurements, the kinetic rate constant (2k(d)) of the bimolecular self-reaction of α-tocopheroxyl radicals in micelles and in human plasma was calculated to be of the order of 10(5) M(-1) s(-1) at 37 °C. This value was obtained considering that the reactive radicals are confined into the micellar pseudophase and is one to two orders of magnitude higher than the value we found in homogeneous phase. The physiological significance of this high value is discussed considering the competition between bimolecular self-reaction and the α-tocopheroxyl radical recycling by ascorbate.

Wild Mediterranean plants as traditional food: a valuable source of antioxidants.

Some wild Mediterranean plants used as traditional food are an extraordinary source of antioxidants. We tested some properties of 10 of these herbaceous plants, used in Liguria (Northwest Italy) to prepare a traditional dish known as "prebuggiun." A total of 9 of them were found to have a polyphenol content and antioxidant properties similar or better than those of red chicory and blueberry, which are, in the case of vegetables and fruits, among the richest of antioxidants. Practical Application: In this article, we reported a study on wild plants growing in the Mediterranean area. These herbs have been neglected and this study aimed to revalue these plants because they are an extraordinary source of antioxidants. The increasing demand for natural antioxidants (additives in the food industry too) justifies the search for new sources of natural antioxidants. The revaluation of these plants will be interesting for: (1) consumer health, rediscovering a vegetable source of high antioxidant power; (2) possibility of producing new commercial products, such as food supplements of high quality and low cost; (3) pharmacological applications.

A method to evaluate capacity and efficiency of water soluble antioxidants as peroxyl radical scavengers.

In this paper, we report on a method to evaluate the activity of water soluble and H-atom donor antioxidants as peroxyl radical scavengers in a micelle system reproducing the conditions occurring in the upper small intestine in humans, during digestion and absorption of lipids. This method, which overcomes some of the problems of the total radical trapping antioxidant parameter (TRAP) assays, measures the peroxyl radical trapping capacity (n) and the peroxyl radical trapping efficiency IC50(-1) of antioxidants, that is the number "n" of peroxyl radicals trapped by one molecule of the studied antioxidant and the reciprocal of the antioxidant concentration that halves the steady-state concentration of peroxyl radicals, respectively. These two fundamental parameters characterizing the radical chain breaking of many water soluble antioxidants, among which dietary polyphenols, can be obtained with relatively good precision from a single experiment, on the basis of a rigorous treatment of the kinetic data.

Synergistic antioxidant effect of catechin and malvidin 3-glucoside on free radical-initiated peroxidation of linoleic acid in micelles.

The inhibitory effect of anthocyanins has been investigated in the peroxidation of linoleic acid in micelles in the presence and in the absence of (+)-catechin. The peroxidation was initiated by thermal decomposition of 2,2(')-azobis[2-(2-imidazolin-2-yl)propane], and the kinetics of peroxidation were followed by measuring the rate of oxygen consumption and the rate of disappearance of the antioxidant. The analysis of the antioxidant effect of various anthocyanins, alone or in the presence of catechin, demonstrates that catechin, which is relatively inefficient at inhibiting linoleic acid oxidation, regenerates the highly efficient antioxidant malvidin 3-glucoside and, at a lower extent, peonidin 3-glucoside. The malvidin 3-glucoside recycling by catechin strongly increases the antioxidant efficiency of these two antioxidants. This protective mechanism appears specific for malvidin and peonidin 3-glucosides. The high unpaired spin density of the phenolic O atoms in the radicals generated by these anthocyanins, calculated by the semiempirical quantum chemical AM1 method, may explain the observed behavior.