The Ionophoric Activity of a Pro-Apoptotic VEGF165 Fragment on HUVEC Cells.

Copper plays an important role as a regulator in many pathologies involving the angiogenesis process. In cancerogenesis, tumor progression, and angiogenic diseases, copper homeostasis is altered. Although many details in the pathways involved are still unknown, some copper-specific ligands have been successfully used as therapeutic agents. Copper-binding peptides able to modulate angiogenesis represent a possible way to value new drugs. We previously reported that a fragment (VEGF73-101) of vascular endothelial growth factor (VEGF165), a potent angiogenic, induced an apoptotic effect on human umbilical vein endothelial cells. The aim of this study was to investigate the putative copper ionophoric activity of VEGF73-101, as well as establish a relationship between the structure of the peptide fragment and the cytotoxic activity in the presence of copper(II) ions. Here, we studied the stoichiometry and the conformation of the VEGF73-101/Cu(II) complexes and some of its mutated peptides by electrospray ionization mass spectrometry and circular dichroism spectroscopy. Furthermore, we evaluated the effect of all peptides in the absence and presence of copper ions by cell viability and cytofuorimetric assays. The obtained results suggest that VEGF73-101 could be considered an interesting candidate in the development of new molecules with ionophoric properties as agents in antiangiogenic therapeutic approaches.

α-Synuclein Aggregated with Tau and ß-Amyloid in Human Platelets from Healthy Subjects: Correlation with Physical Exercise.

The loss of protein homeostasis that has been associated with aging leads to altered levels and conformational instability of proteins, which tend to form toxic aggregates. In particular, brain aging presents characteristic patterns of misfolded oligomers, primarily constituted of ß-amyloid (Aß), tau, and α-synuclein (α-syn), which can accumulate in neuronal membranes or extracellular compartments. Such aging-related proteins can also reach peripheral compartments, thus suggesting the possibility to monitor their accumulation in more accessible fluids. In this respect, we have demonstrated that α-syn forms detectable hetero-aggregates with Aß or tau in red blood cells (RBCs) of healthy subjects. In particular, α-syn levels and its heteromeric interactions are modulated by plasma antioxidant capability (AOC), which increases in turn with physical activity. In order to understand if a specific distribution of misfolded proteins can occur in other blood cells, a cohort of human subjects was enrolled to establish a correlation among AOC, the level of physical exercise and the concentrations of aging-related proteins in platelets. The healthy subjects were divided depending on their level of physical exercise (i.e., athletes and sedentary subjects) and their age (young and older subjects). Herein, aging-related proteins (i.e., α-syn, tau and Aß) were confirmed to be present in human platelets. Among such proteins, platelet tau concentration was demonstrated to decrease in athletes, while α-syn and Aß did not correlate with physical exercise. For the first time, α-syn was shown to directly interact with Aß and tau in platelets, forming detectable hetero-complexes. Interestingly, α-syn interaction with tau was inversely related to plasma AOC and to the level of physical activity. These results suggested that α-syn heterocomplexes, particularly with tau, could represent novel indicators to monitor aging-related proteins in platelets.

The A2B Adenosine Receptor Modulates the Epithelial- Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells.

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-ß1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-ß1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies.

Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells.

The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies.

The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination.

Oxysterols act as promiscuous ligands of class-A GPCRs: in silico molecular modeling and in vitro validation.

According to classical pharmacology, each neurotransmitter/hormonal receptor, including GPCRs, is exclusively activated by highly specific ligands. However, recent evidence challenges this dogma. Oxysterols are produced at inflammatory sites and can surprisingly potently activate the Epstein Barr virus induced gene receptor-2 (EBI2), a GPCR involved in adaptive immune responses. Similarly, oxysterols promiscuously operate CXCR2, a chemokine receptor participating to immune reactions and cancer development. Both EBI2 and CXCR2 are phylogenetically related to GPR17, another GPCR implicated in inflammatory/immune neurodegenerative events. Here, we used an integrated approach combining comparative modeling, molecular docking and in vitro experiments to investigate their potential interactions with oxysterols. All three receptors share the binding site to allocate oxysterols with different local arrangements, higher sensitivity to specific oxysterols and different activation thresholds. Such differences may dictate the diverse biological effects induced by oxysterols, depending on production site, concentration, specific spatiotemporal features and receptor expression on targeted cells. Thus, EBI2, CXCR2 and GPR17 are promiscuously operated by oxysterols making this class of ligands a 'fil rouge' linking oxidative stress, inflammation and neurodegeneration. Such a transversal role may represent a conserved, "unspecific" (but selective) signaling mode, by which emergency molecules activate multiple receptors involved in inflammatory/immune responses.

Combining galantamine and memantine in multitargeted, new chemical entities potentially useful in Alzheimer's disease.

Herein we report on a novel series of multitargeted compounds obtained by linking together galantamine and memantine. The compounds were designed by taking advantage of the crystal structures of acetylcholinesterase (AChE) in complex with galantamine derivatives. Sixteen novel derivatives were synthesized, using spacers of different lengths and chemical composition. The molecules were then tested as inhibitors of AChE and as binders of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Some of the new compounds were nanomolar inhibitors of AChE and showed micromolar affinities for NMDAR. All compounds were also tested for selectivity toward NMDAR containing the 2B subunit (NR2B). Some of the new derivatives showed a micromolar affinity for NR2B. Finally, selected compounds were tested using a cell-based assay to measure their neuroprotective activity. Three of them showed a remarkable neuroprotective profile, inhibiting the NMDA-induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC(50) = 0.28 nM).

In silico identification of new ligands for GPR17: a promising therapeutic target for neurodegenerative diseases.

GPR17, a previously orphan receptor responding to both uracil nucleotides and cysteinyl-leukotrienes, has been proposed as a novel promising target for human neurodegenerative diseases. Here, in order to specifically identify novel potent ligands of GPR17, we first modeled in silico the receptor by using a multiple template approach, in which extracellular loops of the receptor, quite complex to treat, were modeled making reference to the most similar parts of all the class-A GPCRs crystallized so far. A high-throughput virtual screening exploration of GPR17 binding site with more than 130,000 lead-like compounds was then applied, followed by the wet functional and pharmacological validation of the top-scoring chemical structures. This approach revealed successful for the proposed aim, and allowed us to identify five agonists or partial agonists with very diverse chemical structure. None of these compounds could have been expected 'a priori' to act on a GPCR, and all of them behaved as much more potent ligands than GPR17 endogenous activators.

Regulation of A(2A) adenosine receptor expression and functioning following permanent focal ischemia in rat brain.

Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine-mediated-cellular responses. In the present study, we investigated the modulation of rat A(2A) receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A(2A) receptor binding and functioning. On the contrary, in striatum, a significant decrease in A(2A) ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A(2A) receptor-G protein coupling. On the contrary, A(2A) receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up-regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A(2A) ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non-ischemic hemisphere and in sham-operated rats A(2A) ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time-dependent protective effects of specific A(2A) active compounds.

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells.

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

A(2A) adenosine receptor ligands and proinflammatory cytokines induce PC 12 cell death through apoptosis.

A(2A) adenosine receptor-mediated signaling affects a variety of important processes in the central nervous system both in physiological and pathological conditions, and has been indicated as possible novel therapeutic target in several nervous system diseases. In the present work, cell death induction was investigated after neuronal PC 12 cell treatment with proinflammatory cytokines and adenosine receptor ligands. Interleukin-1-beta (IL-1-beta, 500 U/mL), tumor necrosis factor-alpha (TNF-alpha, 1000 U/mL) and the non selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), caused a significant reduction of cell viability with a maximal effect within 3-48 hr. Moreover, an addictive effect was detected when the cells were simultaneously treated with Interleukin-1-beta and NECA for 3 hr. To investigate the adenosine receptor subtypes involved in PC 12 cell death, the effects of several adenosine receptor agonists/antagonists were evaluated. The endogenous nucleoside, adenosine, and the selective A(2A) adenosine receptor agonist, 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide (CGS21680) reduced PC 12 cell viability. This effect was counteracted by the selective A(2A) adenosine receptor antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5c]pyrimidine (SCH58261), but not by selective A(2B) adenosine receptor antagonist N-(4-acethylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706), suggesting the specific involvement of A(2A) adenosine receptor subtype in adenosine-mediated cytotoxicity. Moreover, the selective A(1) adenosine receptor agonist, N(6)-cyclohexyladenosine (CHA), did not induce any significant effect on cell viability. By ELISA immunoassay cell death detection and transmission electron microscopy (TEM) we demonstrated that A(2A) adenosine receptor ligands and cytokines induced cell death through an apoptotic pathway. In conclusion, our results showed that A(2A) adenosine receptors are involved in the control of PC 12 cell survival/death and may contribute to modulate cellular activity in response to tissue damage associated with inflammatory mediator production.

Up-regulation of A(2A) adenosine receptors by proinflammatory cytokines in rat PC12 cells.

The purpose of this study was to examine the regulation of A(2A) adenosine receptor (A(2A) AR) gene expression induced by proinflammatory cytokines in PC12 cells. The A(2A) AR mRNA levels were substantially increased following 3-48 hr PC12 cell treatment with interleukin 1 beta (500 unit/mL) or tumor necrosis factor alpha (1000 unit/mL), as revealed by RT-PCR analysis. In parallel, cell cytokine treatment induced an up-regulation of A(2A) receptor protein. Equilibrium radioligand binding studies on treated-cells showed a significant increase in maximum density of [3H] 2-(carboxyethylphenylethylamino) adenosine-5'-carboxamide binding sites, with no significant changes in the affinity constant value. The increase in A(2A) receptor density was also demonstrated by Western blot analysis. Interleukin 1 beta and tumor necrosis factor alpha effects on A(2A) AR mRNA and protein levels were detectable after 3 hr cytokine treatment and reached a maximum within 24 and 48 hr, respectively. These results demonstrated the existence of heterologous regulation of A(2A) ARs by proinflammatory cytokines. The biological significance of this regulation might be associated with modulating cellular activity in response to tissue damage associated with inflammatory mediator production.