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Nitric oxide is a ubiquitous signaling radical that influences critical body functions. Its importance in the cardiovascular system and the innate immune response to bacterial and viral infections has been extensively investigated. The overproduction of NO is an early component of viral infections, including those affecting the respiratory tract. The production of high levels of NO is due to the overexpression of NO biosynthesis by inducible NO synthase (iNOS), which is involved in viral clearance. The development of NO-based antiviral therapies, particularly gaseous NO inhalation and NO-donors, has proven to be an excellent antiviral therapeutic strategy. The aim of this review is to systematically examine the multiple research studies that have been carried out to elucidate the role of NO in viral infections and to comprehensively describe the NO-based antiviral strategies that have been developed thus far. Particular attention has been paid to the potential mechanisms of NO and its clinical use in the prevention and therapy of COVID-19.

The Guest Editors Federica Sodano, Elena Gazzano, and Roberta Fruttero are pleased to present this editorial overview of the Special Issue entitled “Nitric Oxide Donors for Biomedical Applications: A Themed Issue Dedicated to Professor Alberto Gasco” [...]

Doxorubicin is one of the most effective drugs for the first-line treatment of high-grade osteosarcoma. Several studies have demonstrated that the major cause for doxorubicin resistance in osteosarcoma is the increased expression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). We recently identified a library of H2S-releasing doxorubicins (Sdox) that were more effective than doxorubicin against resistant osteosarcoma cells. Here we investigated the molecular mechanisms of the higher efficacy of Sdox in human osteosarcoma cells with increasing resistance to doxorubicin. Differently from doxorubicin, Sdox preferentially accumulated within the endoplasmic reticulum (ER), and its accumulation was only modestly reduced in Pgp-expressing osteosarcoma cells. The increase in doxorubicin resistance was paralleled by the progressive down-regulation of genes of ER-associated protein degradation/ER-quality control (ERAD/ERQC), two processes that remove misfolded proteins and protect cell from ER stress-triggered apoptosis. Sdox, that sulfhydrated ER-associated proteins and promoted their subsequent ubiquitination, up-regulated ERAD/ERQC genes. This up-regulation, however, was insufficient to protect cells, since Sdox activated ER stress-dependent apoptotic pathways, e.g., the C/EBP-β LIP/CHOP/PUMA/caspases 12-7-3 axis. Sdox also promoted the sulfhydration of Pgp that was subsequently ubiquitinated: this process further enhanced Sdox retention and toxicity in resistant cells. Our work suggests that Sdox overcomes doxorubicin resistance in osteosarcoma cells by at least two mechanisms: it induces the degradation of Pgp following its sulfhydration and produces a huge misfolding of ER-associated proteins, triggering ER-dependent apoptosis. Sdox may represent the prototype of innovative anthracyclines, effective against doxorubicin-resistant/Pgp-expressing osteosarcoma cells by perturbing the ER functions.

Physiologically, smooth muscle cells (SMC) and nitric oxide (NO) produced by endothelial cells strictly cooperate to maintain vasal homeostasis. In atherosclerosis, where this equilibrium is altered, molecules providing exogenous NO and able to inhibit SMC proliferation may represent valuable antiatherosclerotic agents. Searching for dual antiproliferative and NO-donor molecules, we found that furoxans significantly decreased SMC proliferation in vitro, albeit with different potencies. We therefore assessed whether this property is dependent on their thiol-induced ring opening. Indeed, while furazans (analogues unable to release NO) are not effective, furoxans’ inhibitory potency parallels with the electron-attractor capacity of the group in 3 of the ring, making this effect tunable. To demonstrate whether their specific block on G1-S phase could be NO-dependent, we supplemented SMCs with furoxans and inhibitors of GMP- and/or of the polyamine pathway, which regulate NO-induced SMC proliferation, but they failed in preventing the antiproliferative effect. To find the real mechanism of this property, our proteomics studies revealed that eleven cellular proteins (with SUMO1 being central) and networks involved in cell homeostasis/proliferation are modulated by furoxans, probably by interaction with adducts generated after degradation. Altogether, thanks to their dual effect and pharmacological flexibility, furoxans may be evaluated in the future as antiatherosclerotic molecules.

P-glycoprotein (Pgp) determines resistance to a broad spectrum of drugs used against glioblastoma multiforme (GB). Indeed, Pgp is highly expressed in GB stem cells and in the brain-blood barrier (BBB), the peculiar endothelium surrounding the brain. Inhibiting Pgp activity in the BBB and GB is still an open challenge. Here, we tested the efficacy of a small library of tetrahydroisoquinoline derivatives with an EC50 for Pgp ≤ 50 nM, in primary human BBB cells and in patient-derived GB samples, from which we isolated differentiated/adherent cells (AC, i.e., Pgp-negative/doxorubicin-sensitive cells) and stem cells (neurospheres, NS, i.e., Pgp-positive/doxorubicin-resistant cells). Three compounds used at 1 nM increased the delivery of doxorubicin, a typical substrate of Pgp, across BBB monolayer, without altering the expression and activity of other transporters. The compounds increased the drug accumulation within NS, restoring doxorubicin-induced necrosis and apoptosis, and reducing cell viability. In co-culture systems, the compounds added to the luminal face of BBB increased the delivery of doxorubicin to NS growing under BBB and rescued the drug’s cytotoxicity. Our work identified new ligands of Pgp active at low nanomolar concentrations. These compounds reduce Pgp activity in BBB and GB and improve in vitro chemotherapy efficacy in this tumor.

The engineering of photosensitizers (PS) for photodynamic therapy (PDT) with nitric oxide (NO) photodonors (NOPD) is broadening the horizons for new and yet to be fully explored unconventional anticancer treatment modalities that are entirely controlled by light stimuli. In this work, we report a tailored boron-dipyrromethene (BODIPY) derivative that acts as a PS and a NOPD simultaneously upon single photon excitation with highly biocompatible green light. The photogeneration of the two key species for PDT and NOPDT, singlet oxygen (1O2) and NO, has been demonstrated by their direct detection, while the formation of NO is shown not to be dependent on the presence of oxygen. Biological studies carried out using A375 and SKMEL28 cancer cell lines, with the aid of suitable model compounds that are based on the same BODIPY light harvesting core, unambiguously reveal the combined action of 1O2 and NO in inducing amplified cancer cell mortality exclusively under irradiation with visible green light.

Edaravone (5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one, EDV) is a free-radical scavenger reduces organ ischemic injury. Here we investigated whether the protective effects of EDV in renal ischemia/reperfusion (I/R) injury may be enhanced by an EDV derivative bearing a nitric oxide- (NO-) donor furoxan moiety (NO-EDV). Male Wistar rats were subjected to renal ischemia (45 minutes), followed by reperfusion (6 hours). Administration of either EDV (1.2–6–30 µmol/kg, i.v.) or NO-EDV (0.3–1.2–6 µmol/kg, i.v.) dose-dependently attenuated markers of renal dysfunction (serum urea and creatinine, creatinine clearance, urine flow, urinary N-acetyl-β-D-glucosaminidase, and neutrophil gelatinase-associated lipocalin/lipocalin-2). NO-EDV exerted protective effects in the dose-range 1.2–6 µmol/kg, while a higher dose (30 µmol/kg) was needed to obtain protection by EDV. Both EDV and NO-EDV modulated tissue markers of oxidative stress and lipid peroxidation. NO-EDV, but not EDV, activated endothelial NO synthase (NOS) and blunted I/R-induced upregulation of inducible NOS, secondary to modulation of Akt and NF-κB activation, respectively. Besides NO-EDV administration inhibited I/R-induced IL-1β, IL-18, IL-6, and TNF-α overproduction. Overall, these findings demonstrate that the NO-donor moiety contributes to the protection against early renal I/R injury and suggest that NO-donor EDV codrugs are worthy of additional study as innovative pharmacological tools.

Dihydroorotate dehydrogenase (DHODH) is an important drug target due to its prominent role in pyrimidine biosynthesis. Leflunomide and brequinar are two well-known DHODH inhibitors, which bind to the enzyme in the same pocket with different binding modes. We have recently realized a series of new inhibitors based on the 4 - hydroxy - 1,2,5 - oxadiazole ring, whose activity profile was found to be closely dependent on the degree of fluorine substitution at the phenyl ring adjacent to the oxadiazole moiety; a positive influence of fluorine on the DHODH inhibitory potency was observed previously [Baumgartner et al. ( 2006 ) J Med Chem 49:1239–1247]. Potential energy surface scans showed that fluorine plays an important role in stabilizing the bioactive conformations; additionally, fluorine influences the balance between leflunomide-like and brequinar-like binding modes. These findings may serve as a guide to design more potent DHODH inhibitors.

Objectives(1) To evaluate drug–drug interactions (DDIs) in general practitioners’ (GPs) prescriptions; (2) to implement a cooperation project between pharmacists and GPs to improve DDI management and patient care.MethodsIn 2013, pharmacists from the Community Drug Assistance ASL TO1 launched a cooperation project involving 48 GPs. As a first step, GPs were asked to select, from a list, drug associations for which they recommended analysis of occurrence in their prescriptions. The pharmacists (1) analysed GPs’ prescriptions dated 2012–2014, according to the list of DDIs selected (n= 9); (2) evaluated solutions for DDI management, using the Micromedex DDI checker database and literature analysis; they then (3) disseminated DDI-related information to GPs through training meetings and (4) assessed the efficacy of these actions through a questionnaire submitted to the GPs in 2013.Results(1) Prescriptions analysis: a reduction in the number of DDIs was observed (−14% in 2013 vs 2012, –9% in 2014 vs 2012); in some cases these reductions were statistically significant (calcium carbonate + proton pump inhibitors (PPIs) −50%, p<0.0041, amoxicillin+lansoprazole −42%, p<0.0088). (2) Questionnaire: this was completed by 75% of GPs. The literature analysis was considered interesting by 94% of GPs; solutions were adopted by 89% of GPs and 34% of GPs affirmed that clinical improvements after application of the measures were observed in their patients, even if they could not provide quantitative data for this outcome.ConclusionThe cooperation project between pharmacists and GPs was effective because it established a professional exchange between the two health professionals. The pharmacist gave support to GPs, which benefited the patients, who gained clinical improvements and improved satisfaction with their medical care, as declared by the GPs in answers to the questionnaire.

1 Incorporation of a nitric oxide (NO)‐releasing moiety in aspirin can overcome its gastric side effects. 2 We investigated the NO‐release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. 3 Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance‐based technique. Concentration–response curves for antiplatelet agents±the soluble guanylate cyclase inhibitor, ODQ (50 μM) were generated in platelet‐rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. 4 The furoxan derivatives of aspirin (B8, B7) and their NO‐free furazan equivalents (B16, B15; all 100 μM) significantly inhibited COX activity (P<0.01; n=6) in vitro and caused aspirin‐independent, cGMP‐dependent inhibition of collagen‐induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC50=0.62±0.1 μM for B8; 400±89 μM for B7; P<0.0001. WP IC50s=0.6±0.1 and 62±10 μM, respectively). The NO‐free furazan counterparts were less potent antiplatelet agents (WP IC50s=54±3 μM and 62±10 μM, respectively; P<0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP. 5 NO release from furoxan–aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 μM) and ascorbate (50 μM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). 6 We conclude that the decomposition of furoxan–aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition. British Journal of Pharmacology (2006) 148, 517–526. doi:10.1038/sj.bjp.0706743