Alberto Gasco

Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells

Blocking CCL2 nitration in tumors promoted CD8+ influx and reduced tumor growth and prolonged survival in mice when combined with adoptive cell therapy.

A new class of furoxan derivatives as NO donors: mechanism of action and biological activity

1 The mechanism of action and biological activity of a series of R‐substituted and di‐R‐substituted phenylfuroxans is reported. 2 Maximal potency as vasodilators on rabbit aortic rings, precontracted with noradrenaline (1 μm), was shown by phenyl‐cyano isomers and by the 3,4‐dicyanofuroxan, characterized by a potency ratio 3–10 fold higher than glyceryl trinitrate (GTN). This effect was reduced upon coincubation with methylene blue or oxyhaemoglobin (10 μm). 3 The furoxan derivatives showing maximal potency as vasodilators were also able to inhibit collagen‐induced platelet aggregation, with IC50 values in the sub‐micromolar range. 4 The furoxan derivatives were able to stimulate partially purified, rat lung soluble guanylate cyclase; among the most active compounds, the 3‐R‐substituted isomers displayed a higher level of stimulatory effect than the 4‐R analogues. 5 Solutions (0.1 mm) of all the tested furoxans, prepared using 50 mm phosphate buffer, pH 7.4, (diluting 10 mm DMSO stock solutions) did not release nitric oxide (NO) spontaneously; however in presence of 5 mm l‐cysteine, a significant NO‐releasing capacity was observed, which correlated significantly with their stimulation of the guanylate cyclase activity.

N-Substituted analogues of S-nitroso-N-acetyl-D,L-penicillamine: chemical stability and prolonged nitric oxide mediated vasodilatation in isolated rat femoral arteries

Previous studies show that linking acetylated glucosamine to S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium‐denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side‐chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N‐substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S‐nitrosothiols. Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37°C. Bolus injections of SNAP (10 μl; 10−8–10−3 M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400–500 g), caused concentration‐dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium‐intact vessels but failed to recover to pre‐injection pressures at moderate and high concentrations (10−6–10−3 M) in those denuded of endothelium. This sustained effect (>1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin. We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium‐denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage.

NO donors: Focus on furoxan derivatives

The article focuses attention on furoxan derivatives (1,2,5 oxadiazole 2-oxides) as NO donors. Possible mechanisms for NO release from these products in physiological solution and in segments of rabbit femoral artery are briefly considered. The in vitro anti aggregatory activities and the in vitro and in vivo vasodilating properties of a number of furoxans are examined with particular reference to involvement of NO in these actions. The use of the furoxan system to design new NO donor/drug hybrids is discussed in connection with the problem of the balance between NO- and drug dependent activities of the resulting structures. Whether other biological activities (as yet, little studied) of furoxans, such as their antiparasite, antimicrobial, and antitumoral effects, are NO dependent, is a matter still to be explored.

Phenylsulfonylfuroxans as Modulators of Multidrug-Resistance-Associated Protein-1 and P-Glycoprotein

A series of furoxan derivatives were studied for their ability to interact with P-gp and MRP1 transporters in MDCK cells overexpressing these proteins. 3-Phenylsulfonyl substituted furoxans emerged as the most interesting compounds. All of them were capable of inhibiting P-gp, and a few also were capable of inhibiting MRP1. Substituents at the 4-position of 3-phenylsulfonylfuroxan scaffold were able to modulate the selectivity and the intensity of inhibition. In some cases, they reverted MRP1 inhibitor activity, namely, they were capable of potentiating MRP1 dependent efflux. When compounds 16 and 17 were coadministered with doxorubicin, they restored a high degree of the activity of the antibiotic. Preliminary immunoblotting studies carried out on these two compounds indicate that they are capable of nitrating P-gp, which in this form is likely unable to efflux the antibiotic.

Searching for new NO-donor Aspirin-like molecules: a new class of nitrooxy-acyl derivatives of salicylic acid

A new class of products in which the phenol group of salicylic acid is linked to alkanoyl moieties bearing nitrooxy functions has been synthesized and studied for their polyvalent actions. The products were stable in acid and neutral media, while they were hydrolyzed in human serum. Their half-lives were dependent upon the structure of alkanoyl moieties. The products showed anti-inflammatory activities similar to aspirin when tested in the carrageenan-induced paw edema assay in the rat. Interestingly, unlike aspirin, they showed reduced or no gastrotoxicity in a lesion model in rats at equimolar doses. A number of them were able to inhibit platelet aggregation induced by collagen in human platelet-rich plasma. All of the products were capable of relaxing rat aortic strips precontracted with phenylephrine in a concentration-dependent manner. Selected members of this new class of nonsteroidal anti-inflammatory drugs might represent possible safer alternatives to aspirin in different clinical settings.

Mechanisms of Liposomes/Water Partitioning of (p-Methylbenzyl)alkylamines

AbstractPurpose. The objective of this study was to compare and interpret the variations in lipophilicity of homologous (p-methylbenzyl)alkylamines (MBAAs) in isotropic (octanol/water) and anisotropic (zwitterionic liposomes/water) system. Methods. Two experimental approaches were used, namely the pH-metric method to measure lipophilicity parameters in octanol/water and liposomes/water systems, and changes in NMR relaxation rates to validate the former method and to gain additional insights into the mechanisms of liposomes/water partitioning. Results. For long-chain homologues (N-butyl to N-heptyl), the octanol/ water and liposomes/water systems mostly expressed hydrophobicity. In contrast, the lipophilicity of the shorter homologues (N-methyl to N-propyl) in the two systems expressed various electrostatic and polar interactions. Conclusions. The study sheds light on the molecular interactions between zwitterionic liposomes and amphiphilic solutes in neutral and cationic form.

Nitric oxide donor doxorubicins accumulate into Doxorubicin-resistant human colon cancer cells inducing cytotoxicity.

Products 4 and 5, obtained by conjugation of doxorubicin with nitric oxide (NO) donor nitrooxy and phenylsulfonyl furoxan moieties, respectively, accumulate in doxorubicin-resistant human colon cancer cells (HT29-dx), inducing high cytotoxicity. This behavior parallels the ability of the compounds to generate NO, detected as nitrite, in these cells. Preliminary immunoblotting studies suggest that the mechanism that underlies the cytotoxic effect could involve inhibition of cellular drug efflux due to nitration of tyrosine residues of the MRP3 protein pump.

NO donor and biological properties of different benzofuroxans.

AbstractPurpose. To investigate the effect of benzofusion on NO donor properties and related biological activities of the furoxan system. The biological properties considered were the ability to increase the cytosolic levels of cGMP in C6 cells and vasodilation. Methods. NO donor properties were investigated either in the presence or the absence of cysteine by using the Griess reaction, chemiluminescence, and gas chromatography. Increase of cytosolic cGMP levels were evaluated by radioimmunoassay. Vasodilating activity was assessed on rat aorta strips precontracted with noradrenaline, in the presence and the absence of oxyhemoglobin (HbO2) and methylene blue (MB), respectively. Results. Benzofuroxan and its methyl and cyano derivatives were unable to release NO under the experimental conditions. Generally these compounds displayed feeble vasodilating properties and were able to weakly stimulate soluble guanylate cyclase (sGC). By contrast, benzodifuroxan and benzotrifuroxan were able to produce both NO• and its reduced form NO−, the nitroxyl anion. They displayed potent vasodilating properties and were able to increase cytosolic levels of cGMP in a concentration-dependent manner. Conclusions. The simple benzofuroxans considered here are devoid of the capability to release NO, they weakly stimulate sGC as well as manifest feeble vasodilating properties by a mechanism that does not involve a thiol-induced NO production. By contrast, benzodifuroxan and benzotrifuroxan behave as typical NO donor furoxans.

A novel hybrid aspirin-NO-releasing compound inhibits TNFalpha release from LPS-activated human monocytes and macrophages

BackgroundThe cytoprotective nature of nitric oxide (NO) led to development of NO-aspirins in the hope of overcoming the gastric side-effects of aspirin. However, the NO moiety gives these hybrids potential for actions further to their aspirin-mediated anti-platelet and anti-inflammatory effects. Having previously shown that novel NO-aspirin hybrids containing a furoxan NO-releasing group have potent anti-platelet effects, here we investigate their anti-inflammatory properties. Here we examine their effects upon TNFα release from lipopolysaccharide (LPS)-stimulated human monocytes and monocyte-derived macrophages and investigate a potential mechanism of action through effects on LPS-stimulated nuclear factor-kappa B (NF-κB) activation.MethodsPeripheral venous blood was drawn from the antecubital fossa of human volunteers. Mononuclear cells were isolated and cultured. The resultant differentiated macrophages were treated with pharmacologically relevant concentrations of either a furoxan-aspirin (B8, B7; 10 μM), their respective furazan NO-free counterparts (B16, B15; 10 μM), aspirin (10 μM), existing nitroaspirin (NCX4016; 10 μM), an NO donor (DEA/NO; 10 μM) or dexamethasone (1 μM), in the presence and absence of LPS (10 ng/ml; 4 h). Parallel experiments were conducted on undifferentiated fresh monocytes. Supernatants were assessed by specific ELISA for TNFα release and by lactate dehydrogenase (LDH) assay for cell necrosis. To assess NF-κB activation, the effects of the compounds on the loss of cytoplasmic inhibitor of NF-κB, IκBα (assessed by western blotting) and nuclear localisation (assessed by immunofluorescence) of the p65 subunit of NF-κB were determined.ResultsB8 significantly reduced TNFα release from LPS-treated macrophages to 36 ± 10% of the LPS control. B8 and B16 significantly inhibited monocyte TNFα release to 28 ± 5, and 49 ± 9% of control, respectively. The B8 effect was equivalent in magnitude to that of dexamethasone, but was not shared by 10 μM DEA/NO, B7, the furazans, aspirin or NCX4016. LDH assessment revealed none of the treatments caused significant cell lysis. LPS stimulated loss of cytoplasmic IκBα and nuclear translocation of the p65 NF-κB subunit was inhibited by the active NO-furoxans.ConclusionHere we show that furoxan-aspirin, B8, significantly reduces TNFα release from both monocytes and macrophages and suggest that inhibition of NF-κB activation is a likely mechanism for the effect. This anti-inflammatory action highlights a further therapeutic potential of drugs of this class.