Publications by authors named "Maria Letizia Trincavelli"

71 Publications

Cornus sanguinea Fruits: a Source of Antioxidant and Antisenescence Compounds Acting on Aged Human Dermal and Gingival Fibroblasts.

Planta Med 2021 Apr 15. Epub 2021 Apr 15.

Dipartimento di Farmacia, Università di Pisa, Pisa, Italy.

Five new compounds, a flavonol glycoside (1: ), a megastigmane (2: ), 2 cyclohexylethanoids (3, 4: ), and a phenylethanoid derivative (5: ), together with 15 known compounds (6: -20: ) including flavonoid glycosides, cyclohexylethanoids, and phenolic compounds, have been isolated from drupes. All the structures have been determined by 1D and 2D NMR spectroscopic analysis and mass spectrometry data. The antioxidant capability of the most representative isolated compounds was evaluated in the hydrogen peroxide (HO)-induced premature cellular senescence model of human dermal and gingival fibroblasts. Several derivatives counteracted the increase of reactive oxigen species (ROS) production in both cellular models. Among the most promising, compounds 8, 14: , and 20: were able to counteract cell senescence, decreasing the expression of p21 and p53. Furthermore, compound 14: decreased the expression of inflammatory cytokines (IL-6) in both cell models and counteracted the decrease of collagen expression induced by the HO in dermal human fibroblasts. These data highlight the anti-aging properties of several isolated compounds from drupes, supporting its possible use in the cure of skin or periodontitis lesions.
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http://dx.doi.org/10.1055/a-1471-6666DOI Listing
April 2021

Allosterism vs. Orthosterism: Recent Findings and Future Perspectives on A AR Physio-Pathological Implications.

Front Pharmacol 2021 24;12:652121. Epub 2021 Mar 24.

Department of Pharmacy, University of Pisa, Pisa, Italy.

The development of GPCR (G-coupled protein receptor) allosteric modulators has attracted increasing interest in the last decades. The use of allosteric modulators in therapy offers several advantages with respect to orthosteric ones, as they can fine-tune the tissue responses to the endogenous agonist. Since the discovery of the first A adenosine receptor (AR) allosteric modulator in 1990, several efforts have been made to develop more potent molecules as well as allosteric modulators for all adenosine receptor subtypes. There are four subtypes of AR: A, A, A, and A. Positive allosteric modulators of the A AR have been proposed for the cure of pain. A positive allosteric modulators are thought to be beneficial during inflammatory processes. More recently, A and A AR allosteric modulators have also been disclosed. The A AR displays the lowest affinity for its endogenous ligand adenosine and is mainly activated as a consequence of tissue damage. The A AR activation has been found to play a crucial role in chronic obstructive pulmonary disease, in the protection of the heart from ischemic injury, and in the process of bone formation. In this context, allosteric modulators of the A AR may represent pharmacological tools useful to develop new therapeutic agents. Herein, we provide an up-to-date highlight of the recent findings and future perspectives in the field of orthosteric and allosteric A AR ligands. Furthermore, we compare the use of orthosteric ligands with positive and negative allosteric modulators for the management of different pathological conditions.
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http://dx.doi.org/10.3389/fphar.2021.652121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024542PMC
March 2021

Ruthenium(II) 1,4,7-trithiacyclononane complexes of curcumin and bisdemethoxycurcumin: Synthesis, characterization, and biological activity.

J Inorg Biochem 2021 May 20;218:111387. Epub 2021 Feb 20.

Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, Trieste, Italy.

Two cationic ruthenium(II) 1,4,7-trithiacyclononane ([9]aneS) complexes of curcumin (curcH) and bisdemethoxycurcumin (bdcurcH), namely [Ru(curc)(dmso-S)([9]aneS)]Cl (1) and [Ru(bdcurc)(dmso-S)([9]aneS)]Cl (2) were prepared from the [RuCl(dmso-S)([9]-aneS)] precursor and structurally characterized, both in solution and in the solid state by X-ray crystallography. The corresponding PTA complexes [Ru(curc)(PTA)([9]aneS)]Cl (3) and [Ru(bdcurc)(PTA)([9]aneS)]Cl (4) have been also synthesized and characterized (PTA = 1,3,5-triaza-7-phosphaadamantane). Bioinorganic studies relying on mass spectrometry were performed on complexes 1-4 to assess their interactions with the model protein lysozyme. Overall, a rather limited reactivity with lysozyme was highlighted accompanied by a modest cytotoxic potency against three representative cancer cell lines. The moderate pharmacological activity is likely connected to the relatively high stability of these complexes.
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http://dx.doi.org/10.1016/j.jinorgbio.2021.111387DOI Listing
May 2021

α-Synuclein Heteromers in Red Blood Cells of Alzheimer's Disease and Lewy Body Dementia Patients.

J Alzheimers Dis 2021 ;80(2):885-893

Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.

Background: Red blood cells (RBCs) contain the majority of α-synuclein (α-syn) in blood, representing an interesting model for studying the peripheral pathological alterations proved in neurodegeneration.

Objective: The current study aimed to investigate the diagnostic value of total α-syn, amyloid-β (Aβ1-42), tau, and their heteroaggregates in RBCs of Lewy body dementia (LBD) and Alzheimer's disease (AD) patients compared to healthy controls (HC).

Methods: By the use of enzyme-linked immunosorbent assays, RBCs concentrations of total α-syn, Aβ1-42, tau, and their heteroaggregates (α-syn/Aβ1-42 and α-syn/tau) were measured in 27 individuals with LBD (Parkinson's disease dementia, n = 17; dementia with Lewy bodies, n = 10), 51 individuals with AD (AD dementia, n = 37; prodromal AD, n = 14), and HC (n = 60).

Results: The total α-syn and tau concentrations as well as α-syn/tau heterodimers were significantly lower in the LBD group and the AD group compared with HC, whereas α-syn/Aβ1-42 concentrations were significantly lower in the AD dementia group only. RBC α-syn/tau heterodimers had a higher diagnostic accuracy for differentiating patients with LBD versus HC (AUROC = 0.80).

Conclusion: RBC α-syn heteromers may be useful for differentiating between neurodegenerative dementias (LBD and AD) and HC. In particular, RBC α-syn/tau heterodimers have demonstrated good diagnostic accuracy for differentiating LBD from HC. However, they are not consistently different between LBD and AD. Our findings also suggest that α-syn, Aβ1-42, and tau interact in vivo to promote the aggregation and accumulation of each other.
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http://dx.doi.org/10.3233/JAD-201038DOI Listing
January 2021

Novel positive allosteric modulators of A adenosine receptor acting as bone mineralisation promoters.

J Enzyme Inhib Med Chem 2021 Dec;36(1):286-294

Department of Pharmacy, University of Pisa, Pisa, Italy.

Small-molecules acting as positive allosteric modulators (PAMs) of the A adenosine receptor (A AR) could potentially represent a novel therapeutic strategy for pathological conditions characterised by altered bone homeostasis, including osteoporosis. We investigated a library of compounds (-) exhibiting different degrees of chemical similarity with three indole derivatives (-), which have been recently identified by us as PAMs of the A AR able to promote mesenchymal stem cell differentiation and bone formation. Evaluation of mineralisation activity of - in the presence and in the absence of the agonist BAY60-6583 allowed the identification of lead compounds with therapeutic potential as anti-osteoporosis agents. Further biological characterisation of one of the most performing compounds, the benzofurane derivative , confirmed that such a molecule behaves as PAM of the A AR.
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http://dx.doi.org/10.1080/14756366.2020.1862103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751416PMC
December 2021

High Adenosine Extracellular Levels Induce Glioblastoma Aggressive Traits Modulating the Mesenchymal Stromal Cell Secretome.

Int J Mol Sci 2020 Oct 18;21(20). Epub 2020 Oct 18.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.

Glioblastoma is an aggressive, fast-growing brain tumor influenced by the composition of the tumor microenvironment (TME) in which mesenchymal stromal cell (MSCs) play a pivotal role. Adenosine (ADO), a purinergic signal molecule, can reach up to high micromolar concentrations in TME. The activity of specific adenosine receptor subtypes on glioma cells has been widely explored, as have the effects of MSCs on tumor progression. However, the effects of high levels of ADO on glioma aggressive traits are still unclear as is its role in cancer cells-MSC cross-talk. Herein, we first studied the role of extracellular Adenosine (ADO) on isolated human U343MG cells as a glioblastoma cellular model, finding that at high concentrations it was able to prompt the gene expression of Snail and ZEB1, which regulate the epithelial-mesenchymal transition (EMT) process, even if a complete transition was not reached. These effects were mediated by the induction of ERK1/2 phosphorylation. Additionally, ADO affected isolated bone marrow derived MSCs (BM-MSCs) by modifying the pattern of secreted inflammatory cytokines. Then, the conditioned medium (CM) of BM-MSCs stimulated with ADO and a co-culture system were used to investigate the role of extracellular ADO in GBM-MSC cross-talk. The CM promoted the increase of glioma motility and induced a partial phenotypic change of glioblastoma cells. These effects were maintained when U343MG cells and BM-MSCs were co-cultured. In conclusion, ADO may affect glioma biology directly and through the modulation of the paracrine factors released by MSCs overall promoting a more aggressive phenotype. These results point out the importance to deeply investigate the role of extracellular soluble factors in the glioma cross-talk with other cell types of the TME to better understand its pathological mechanisms.
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http://dx.doi.org/10.3390/ijms21207706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589183PMC
October 2020

Prodromal Intestinal Events in Alzheimer's Disease (AD): Colonic Dysmotility and Inflammation Are Associated with Enteric AD-Related Protein Deposition.

Int J Mol Sci 2020 May 15;21(10). Epub 2020 May 15.

Unit of Pharmacology and Pharmacovigilance, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy.

Increasing evidence suggests that intestinal dysfunctions may represent early events in Alzheimer's disease and contribute to brain pathology. This study examined the relationship between onset of cognitive impairment and colonic dysfunctions in a spontaneous AD model before the full development of brain pathology. SAMP8 mice underwent Morris water maze and assessment of faecal output at four, six and eight months of age. In vitro colonic motility was examined. Faecal and colonic Aβ, tau proteins, α-synuclein and IL-1β were assessed by ELISA. Colonic citrate synthase activity was assessed by spectrophotometry. Colonic NLRP3, caspase-1 and ASC expression were evaluated by Western blotting. Colonic eosinophil density and claudin-1 expression were evaluated by immunohistochemistry. The effect of Aβ on NLRP3 signalling and mitochondrial function was tested in cultured cells. Cognitive impairment and decreased faecal output occurred in SAMP8 mice from six months. When compared with SAMR1, SAMP8 animals displayed: (1) impaired in vitro colonic contractions; (2) increased enteric AD-related proteins, IL-1β, active-caspase-1 expression and eosinophil density; and (3) decreased citrate synthase activity and claudin-1 expression. In THP-1 cells, Aβ promoted IL-1β release, which was abrogated upon incubation with caspase-1 inhibitor or in ASC cells. Aβ decreased mitochondrial function in THP-1 cells. In SAMP8, enteric AD-related proteins deposition, inflammation and impaired colonic excitatory neurotransmission, occurring before the full brain pathology development, could contribute to bowel dysmotility and represent prodromal events in AD.
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http://dx.doi.org/10.3390/ijms21103523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278916PMC
May 2020

Development of the first in vivo GPR17 ligand through an iterative drug discovery pipeline: A novel disease-modifying strategy for multiple sclerosis.

PLoS One 2020 22;15(4):e0231483. Epub 2020 Apr 22.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy.

The GPR17 receptor, expressed on oligodendroglial precursors (OPCs, the myelin producing cells), has emerged as an attractive target for a pro-myelinating strategy in multiple sclerosis (MS). However, the proof-of-concept that selective GPR17 ligands actually exert protective activity in vivo is still missing. Here, we exploited an iterative drug discovery pipeline to prioritize novel and selective GPR17 pro-myelinating agents out of more than 1,000,000 compounds. We first performed an in silico high-throughput screening on GPR17 structural model to identify three chemically-diverse ligand families that were then combinatorially exploded and refined. Top-scoring compounds were sequentially tested on reference pharmacological in vitro assays with increasing complexity, ending with myelinating OPC-neuron co-cultures. Successful ligands were filtered through in silico simulations of metabolism and pharmacokinetics, to select the most promising hits, whose dose and ability to target the central nervous system were then determined in vivo. Finally, we show that, when administered according to a preventive protocol, one of them (named by us as galinex) is able to significantly delay the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. This outcome validates the predictivity of our pipeline to identify novel MS-modifying agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231483PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176092PMC
July 2020

Antioxidant Activity of Compounds Isolated from Promotes Human Gingival Fibroblast Well-Being.

J Nat Prod 2020 03 7;83(3):626-637. Epub 2020 Feb 7.

Dipartimento di Farmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy.

Four new triterpenoid bidesmosidic saponins (-) and a sesquiterpenoid glucoside (), together with nine known phenolic compounds (-), were isolated from the fruits of . Their structures were elucidated using 1D and 2D NMR spectroscopy and mass spectrometry data. The antioxidant capability of the isolated compounds was evaluated in human gingival fibroblasts. Compound decreased ROS production and promoted cell proliferation. It also counteracted the cell cycle blockade induced by a low concentration of HO decreasing the expression of p21 and CDKN2A (p16). Compound decreased the expression of inflammatory cytokines (IL-6 and IL-8) in response to inflammatory stimuli, supporting its possible use in periodontitis lesions.
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http://dx.doi.org/10.1021/acs.jnatprod.9b01030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997630PMC
March 2020

Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination.

Front Chem 2019 10;7:910. Epub 2020 Jan 10.

Department of Biotechnology, University of Verona, Verona, Italy.

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.
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http://dx.doi.org/10.3389/fchem.2019.00910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966494PMC
January 2020

Molecular insight on the altered membrane trafficking of TrkA kinase dead mutants.

Biochim Biophys Acta Mol Cell Res 2020 02 21;1867(2):118614. Epub 2019 Nov 21.

Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy; Department of Pharmacy, University of Pisa, Pisa, Italy. Electronic address:

We address the contribution of kinase domain structure and catalytic activity to membrane trafficking of TrkA receptor tyrosine kinase. We conduct a systematic comparison between TrkA-wt, an ATP-binding defective mutant (TrkA-K544N) and other mutants displaying separate functional impairments of phosphorylation, ubiquitination, or recruitment of intracellular partners. We find that only K544N mutation endows TrkA with restricted membrane mobility and a substantial increase of cell surface pool already in the absence of ligand stimulation. This mutation is predicted to drive a structural destabilization of the αC helix in the N-lobe by molecular dynamics simulations, and enhances interactions with elements of the actin cytoskeleton. On the other hand, a different TrkA membrane immobilization is selectively observed after NGF stimulation, requires both phosphorylation and ubiquitination to occur, and is most probably related to the signaling abilities displayed by the wt but not mutated receptors. In conclusion, our results allow to distinguish two different TrkA membrane immobilization modes and demonstrate that not all kinase-inactive mutants display identical membrane trafficking.
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http://dx.doi.org/10.1016/j.bbamcr.2019.118614DOI Listing
February 2020

High Levels of -Amyloid, Tau, and Phospho-Tau in Red Blood Cells as Biomarkers of Neuropathology in Senescence-Accelerated Mouse.

Oxid Med Cell Longev 2019 9;2019:5030475. Epub 2019 Jun 9.

Department of Pharmacy, University of Pisa, Pisa, Italy.

Alzheimer's Disease (AD) is the most common Neurodegenerative Disease (ND), primarily characterised by neuroinflammation, neuronal plaques of -amyloid (A), and neurofibrillary tangles of hyperphosphorylated tau. -Synuclein (-syn) and its heteroaggregates with A and tau have been recently included among the neuropathological elements of NDs. These pathological traits are not restricted to the brain, but they reach peripheral fluids as well. In this sense, Red Blood Cells (RBCs) are emerging as a good model to investigate the biochemical alterations of aging and NDs. Herein, the levels of homo- and heteroaggregates of ND-related proteins were analysed at different stages of disease progression. In particular, a validated animal model of AD, the SAMP8 (Senescence-Accelerated Mouse-Prone) and its control strain SAMR1 (Senescence-Accelerated Mouse-Resistant) were used in parallel experiments. The levels of the aforementioned proteins and of the inflammatory marker interleukin-1 (IL-1) were examined in both brain and RBCs of SAMP8 and SAMR1 at 6 and 8 months. Brain A, tau, and phospho-tau (p-tau) were higher in SAMP8 mice than in control mice and increased with AD progression. Similar accumulation kinetics were found in RBCs, even if slower. By contrast, -syn and its heterocomplexes (-syn-A and -syn-tau) displayed different accumulation kinetics between brain tissue and RBCs. Both brain and peripheral IL-1 levels were higher in SAMP8 mice, but increased sooner in RBCs, suggesting that inflammation might initiate at a peripheral level before affecting the brain. In conclusion, these results confirm RBCs as a valuable model for monitoring neurodegeneration, suggesting peripheral A, tau, and p-tau as potential early biomarkers of AD.
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http://dx.doi.org/10.1155/2019/5030475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590616PMC
February 2020

Potential Diagnostic Value of Red Blood Cells α-Synuclein Heteroaggregates in Alzheimer's Disease.

Mol Neurobiol 2019 Sep 2;56(9):6451-6459. Epub 2019 Mar 2.

Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy.

A plethora of complex misfolded protein combinations have been found in Alzheimer disease (AD) brains besides the classical pathological hallmarks. Recently, α-synuclein (α-syn) and its heterocomplexes with amyloid-β (Aβ) and tau have been suggested to be involved in the pathophysiological processes of neurodegenerative diseases. These pathological features are not limited to the brain, but can be also found in peripheral fluids. In this respect, red blood cells (RBCs) have been suggested as a good model to investigate the biochemical alterations of neurodegeneration. Our aim is to find whether RBC concentrations of α-syn and its heterocomplexes (i.e., α-syn/Aβ and α-syn/tau) were different in AD patients compared with healthy controls (HC). The levels of homo- and heteroaggregates of α-syn, Aβ and tau, were analyzed in a cohort of AD patients at early stage either with dementia or prodromal symptoms (N = 39) and age-matched healthy controls (N = 39). All AD patients received a biomarker-based diagnosis (low cerebrospinal fluid levels of Aβ peptide combined with high cerebrospinal fluid concentrations of total tau and/or phospho-tau proteins; alternatively, a positivity to cerebral amyloid-PET scan). Our results showed lower concentrations of α-syn and its heterocomplexes (i.e., α-syn/Aβ and α-syn/tau) in RBCs of AD patients with respect to HC. RBC α-syn/Aβ as well as RBC α-syn/tau heterodimers discriminated AD participants from HC with fair accuracy, whereas RBC α-syn concentrations differentiated poorly the two groups. Although additional investigations are required, these data suggest α-syn heteroaggregates in RBCs as potential tool in the diagnostic work-up of early AD diagnosis.
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http://dx.doi.org/10.1007/s12035-019-1531-4DOI Listing
September 2019

Long lasting inhibition of Mdm2-p53 interaction potentiates mesenchymal stem cell differentiation into osteoblasts.

Biochim Biophys Acta Mol Cell Res 2019 05 28;1866(5):737-749. Epub 2019 Jan 28.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.

The osteoblast generation from Mesenchymal stem cells (MSCs) is tightly coordinated by transcriptional networks and signalling pathways that control gene expression and protein stability of osteogenic "master transcription factors". Among these pathways, a great attention has been focused on p53 and its physiological negative regulator, the E3 ligase Murine double minute 2 (Mdm2). Nevertheless, the signalling that regulates Mdm2-p53 axis in osteoblasts remain to be elucidated, also considering that Mdm2 possesses numerous p53-independent activities and interacts with additional proteins. Herein, the effects of Mdm2 modulation on MSC differentiation were examined by the use of short- and long-lasting inhibitors of the Mdm2-p53 complex. The long-lasting Mdm2-p53 dissociation was demonstrated to enhance the MSC differentiation into osteoblasts. The increase of Mdm2 levels promoted its association to G protein-coupled receptors kinase (GRK) 2, one of the most relevant kinases involved in the desensitization of G protein-coupled receptors (GPCRs). In turn, the long-lasting Mdm2-p53 dissociation decreased GRK2 levels and favoured the functionality of A Adenosine Receptors (AARs), a GPCR dictating MSC fate. EB148 facilitated cAMP accumulation, and mediated a sustained activation of extracellular signal-regulated kinases (ERKs) and cAMP response element-binding protein (CREB). Such pro-osteogenic effects were not detectable by using the reversible Mdm2-p53 complex inhibitor, suggesting the time course of Mdm2-p53 dissociation may impact on intracellular proteins involved in cell differentiation fate. These results suggest that the long-lasting Mdm2 binding plays a key role in the mobilization of intracellular proteins that regulate the final biological outcome of MSCs.
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http://dx.doi.org/10.1016/j.bbamcr.2019.01.012DOI Listing
May 2019

Novel fluorescent triazinobenzimidazole derivatives as probes for labelling human A and A adenosine receptor subtypes.

Bioorg Med Chem 2018 12 1;26(22):5885-5895. Epub 2018 Nov 1.

Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, I-56126 Pisa, Italy.

The expression levels and the subcellular localization of adenosine receptors (ARs) are affected in several pathological conditions as a consequence of changes in adenosine release and metabolism. In this respect, labelled probes able to monitor the AR expression could be a useful tool to investigate different pathological conditions. Herein, novel ligands for ARs, bearing the fluorescent 7-nitrobenzofurazan (NBD) group linked to the N (1,2) or N (3,4) nitrogen of a triazinobenzimidazole scaffold, were synthesized. The compounds were biologically evaluated as fluorescent probes for labelling A and A AR subtypes in bone marrow-derived mesenchymal stem cells (BM-MSCs) that express both receptor subtypes. The binding affinity of the synthetized compounds towards the different AR subtypes was determined. The probe 3 revealed a higher affinity to A and A ARs, showing interesting spectroscopic properties, and it was selected as the most suitable candidate to label both AR subtypes in undifferentiated MSCs. Fluorescence confocal microscopy showed that compound 3 significantly labelled ARs on cell membranes and the fluorescence signal was decreased by the cell pre-incubation with the A AR and A AR selective agonists, R-PIA and BAY 60-6583, respectively, thus confirming the specificity of the obtained signal. In conclusion, compound 3 could represent a useful tool to investigate the expression pattern of both A and A ARs in different pathological and physiological processes. Furthermore, these results provide an important basis for the design of new and more selective derivatives able to monitor the expression and localization of each different ARs in several tissues and living cells.
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http://dx.doi.org/10.1016/j.bmc.2018.10.039DOI Listing
December 2018

Studies on enantioselectivity of chiral 4-acetylamino-6-alkyloxy-2-alkylthiopyrimidines acting as antagonists of the human A adenosine receptor.

Medchemcomm 2018 Jan 9;9(1):81-86. Epub 2017 Nov 9.

Dipartimento di Farmacia , Università di Pisa , Via Bonanno Pisano 6 , 56126 Pisa , Italy.

Three A adenosine receptor (AR) antagonists () selected from 4-acylamino-6-alkyloxy-2-alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or thioether side chains. These compounds gave us the chance to evaluate whether their higher lipophilicity, reduced conformational freedom and chirality might improve the potency towards the A AR. Racemic mixtures of were resolved using chiral HPLC methods and the absolute configurations of the enantiomers were assigned by chiroptical spectroscopy and density functional theory calculations. We measured the affinity for human A, A, A and A ARs of the racemic mixtures and the pure enantiomers of by radioligand competition binding experiments. Cell-based assays of the most potent enantiomers confirmed their A AR antagonist profiles. Our research led to the identification of ()- with high potency (0.5 nM) and selectivity as an A AR antagonist. Moreover we built a docking-model useful to design new pyrimidine derivatives.
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http://dx.doi.org/10.1039/c7md00375gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072526PMC
January 2018

Epigenetic Modifications of the -Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects.

Oxid Med Cell Longev 2018 15;2018:3740345. Epub 2018 Apr 15.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.

Epigenetic regulation may contribute to the beneficial effects of physical activity against age-related neurodegeneration. For example, epigenetic alterations of the gene encoding for -synuclein () have been widely explored in both brain and peripheral tissues of Parkinson's disease samples. However, no data are currently available about the effects of physical exercise on epigenetic regulation in ageing healthy subjects. The present paper explored whether, in healthy individuals, age and physical activity are related to blood intron1- ( ) methylation, as well as further parameters linked to such epigenetic modification (total, oligomeric -synuclein and DNA methyltransferase concentrations in the blood). Here, the methylation status increased with ageing, and consistent with this result, low -synuclein levels were found in the blood. The direct relationship between methylation and -synuclein levels was observed in samples characterized by blood -synuclein concentrations of 76.3 ng/mg protein or lower (confidence interval (CI) = 95%). In this selected population, higher physical activity reduced the total and oligomeric -synuclein levels. Taken together, our data shed light on ageing- and physical exercise-induced changes on the methylation status and protein levels of -synuclein.
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http://dx.doi.org/10.1155/2018/3740345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924988PMC
October 2018

α-Synuclein Heterocomplexes with β-Amyloid Are Increased in Red Blood Cells of Parkinson's Disease Patients and Correlate with Disease Severity.

Front Mol Neurosci 2018 22;11:53. Epub 2018 Feb 22.

Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid (Aβ) and tau, in both brain and peripheral tissues. In addition to oligomers, the role of the interactions of α-syn with Aβ or tau has gradually emerged. Nevertheless, despite intensive research, NDs have no accepted peripheral markers for biochemical diagnosis. In this respect, Red Blood Cells (RBCs) are emerging as a valid peripheral model for the study of aging-related pathologies. Herein, a small cohort ( = 28) of patients affected by Parkinson's disease (PD) and age-matched controls were enrolled to detect the content of α-syn (total and oligomeric), Aβ and tau (total and phosphorylated) in RBCs. Moreover, the presence of α-syn association with tau and Aβ was explored by co-immunoprecipitation/western blotting in the same cells, and quantitatively confirmed by immunoenzymatic assays. For the first time, PD patients were demonstrated to exhibit α-syn heterocomplexes with Aβ and tau in peripheral tissues; interestingly, α-syn-Aβ concentrations were increased in PD subjects with respect to healthy controls (HC), and directly correlated with disease severity and motor deficits. Moreover, total-α-syn levels were decreased in PD subjects and inversely related to their motor deficits. Finally, an increase of oligomeric-α-syn and phosphorylated-tau was observed in RBCs of the enrolled patients. The combination of three parameters (total-α-syn, phosphorylated-tau and α-syn-Aβ concentrations) provided the best fitting predictive index for discriminating PD patients from controls. Nevertheless further investigations should be required, overall, these data suggest α-syn hetero-aggregates in RBCs as a putative tool for the diagnosis of PD.
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http://dx.doi.org/10.3389/fnmol.2018.00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827358PMC
February 2018

Dual MET and SMO Negative Modulators Overcome Resistance to EGFR Inhibitors in Human Nonsmall Cell Lung Cancer.

J Med Chem 2017 09 22;60(17):7447-7458. Epub 2017 Aug 22.

DiSTABiF, Università della Campania "Luigi Vanvitelli" , Via Vivaldi 43, 81100 Caserta, Italy.

Tyrosine kinase inhibitors (TKIs) of the EGF receptor (EGFR) have provided a significant improvement in the disease outcome of nonsmall cell lung cancer (NSCLC). Unfortunately, resistance to these agents frequently occurs, and it is often related to the activation of the Hedgehog (Hh) and MET signaling cascades driving the epithelial-to-mesenchymal transition (EMT). Because the concomitant inhibition of both Hh and MET pathways restores the sensitivity to anti-EGFR drugs, here we aimed at discovering the first compounds that block simultaneously MET and SMO. By using an "in silico drug repurposing" approach and by validating our predictions both in vitro and in vivo, we identified a set of compounds with the desired dual inhibitory activity and enhanced antiproliferative activity on EGFR TKI-resistant NSCLC. The identification of the known MET TKIs, glesatinib and foretinib, as negative modulators of the Hh pathway, widens their application in the context of NSCLC.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00794DOI Listing
September 2017

α-Synuclein Aggregates with β-Amyloid or Tau in Human Red Blood Cells: Correlation with Antioxidant Capability and Physical Exercise in Human Healthy Subjects.

Mol Neurobiol 2018 03 18;55(3):2653-2675. Epub 2017 Apr 18.

Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126, Pisa, Italy.

Neurodegenerative disorders (NDs) are characterized by abnormal accumulation/misfolding of specific proteins, primarily α-synuclein (α-syn), β-amyloid (Aβ), and tau, in both brain and peripheral tissue. In addition to homo-oligomers, the role of α-syn interactions with Aβ or tau has gradually emerged. The altered protein accumulation has been related to both oxidative stress and physical activity; nevertheless, no correlation among the presence of peripheral α-syn hetero-aggregates, antioxidant capacity, and physical exercise has been discovered as of yet. Herein, the content of α-syn, Aβ, tau, and of their heterocomplexes was determined in red blood cells (RBCs) of healthy subjects (sedentary and athletes). Such parameters were related to the extent of the antioxidant capability (AOC), a key marker of oxidative stress in aging-related pathologies, and to physical exercise, which is known to play an important preventive role in NDs and to modulate oxidative stress. Tau content and plasma AOC toward hydroxyl radicals were both reduced in older or sedentary subjects; in contrast, α-syn and Aβ accumulated in elderly subjects and showed an inverse correlation with both hydroxyl AOC and the level of physical activity. For the first time, α-syn heterocomplexes with Aβ or tau were quantified and demonstrated to be inversely related to hydroxyl AOC. Furthermore, α-syn/Aβ aggregates were significantly reduced in athletes and inversely correlated with physical activity level, independent of age. The positive correlation between antioxidant capability/physical activity and reduced protein accumulation was confirmed by these data and suggested that peripheral α-syn heterocomplexes may represent new indicators of ND-related protein misfolding.
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http://dx.doi.org/10.1007/s12035-017-0523-5DOI Listing
March 2018

Bax Activation Blocks Self-Renewal and Induces Apoptosis of Human Glioblastoma Stem Cells.

ACS Chem Neurosci 2018 01 11;9(1):85-99. Epub 2017 Apr 11.

Department of Pharmacy, University of Naples Federico II , 80131 Napoli, Italy.

Glioblastoma (GBM) is characterized by a poor response to conventional chemotherapeutic agents, attributed to the insurgence of drug resistance mechanisms and to the presence of a subpopulation of glioma stem cells (GSCs). GBM cells and GSCs present, among others, an overexpression of antiapoptotic proteins and an inhibition of pro-apoptotic ones, which help to escape apoptosis. Among pro-apoptotic inducers, the Bcl-2 family protein Bax has recently emerged as a promising new target in cancer therapy along with first BAX activators (BAM7, Compound 106, and SMBA1). Herein, a derivative of BAM-7, named BTC-8, was employed to explore the effects of Bax activation in different human GBM cells and in their stem cell subpopulation. BTC-8 inhibited GBM cell proliferation, arrested the cell cycle, and induced apoptosis through the induction of mitochondrial membrane permeabilization. Most importantly, BTC-8 blocked proliferation and self-renewal of GSCs and induced their apoptosis. Notably, BTC-8 was demonstrated to sensitize both GBM cells and GSCs to the alkylating agent Temozolomide. Overall, our findings shed light on the effects and the relative molecular mechanisms related to Bax activation in GBM, and they suggest Bax-targeting compounds as promising therapeutic tools against the GSC reservoir.
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http://dx.doi.org/10.1021/acschemneuro.7b00023DOI Listing
January 2018

Osteogenesis Is Improved by Low Tumor Necrosis Factor Alpha Concentration through the Modulation of Gs-Coupled Receptor Signals.

Mol Cell Biol 2017 04 31;37(8). Epub 2017 Mar 31.

Department of Pharmacy, University of Pisa, Pisa, Italy.

In the early phase of bone damage, low concentrations of the cytokine tumor necrosis factor alpha (TNF-α) favor osteoblast differentiation. In contrast, chronic high doses of the same cytokine contribute to bone loss, demonstrating opposite effects depending on its concentration and on the time of exposure. In the bone microenvironment, TNF-α modulates the expression/function of different G protein-coupled receptors (GPCRs) and of their regulatory proteins, GPCR-regulated kinases (GRKs), thus dictating their final biological outcome in controlling bone anabolic processes. Here, the effects of TNF-α were investigated on the expression/responsiveness of the A adenosine receptor (AAR), a Gs-coupled receptor that promotes mesenchymal stem cell (MSC) differentiation into osteoblasts. Low TNF-α concentrations exerted a prodifferentiating effect on MSCs, pushing them toward an osteoblast phenotype. By regulating GRK2 turnover and expression, the cytokine impaired AAR desensitization, accelerating receptor-mediated osteoblast differentiation. These data supported the anabolic effect of TNF-α submaximal concentration and demonstrated that the cytokine regulates GPCR responses by interfering with the receptor desensitization machinery, thereby enhancing the anabolic responses evoked by AAR ligands. Overall, these results indicated that GPCR desensitization plays a pivotal role in osteogenesis and that its manipulation is an effective strategy to favor bone remodeling.
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http://dx.doi.org/10.1128/MCB.00442-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376638PMC
April 2017

4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives as novel non-nucleoside agonists for the adenosine A receptor.

Chem Biol Drug Des 2016 11 11;88(5):724-729. Epub 2016 Jul 11.

Dipartimento di Farmacia, Università di Pisa, Pisa, Italy.

Three 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives (4-6) were investigated as potential non-nucleoside agonists at human adenosine receptors (ARs). When tested in competition binding experiments, these compounds exhibited low micromolar affinity (K values comprised between 1.2 and 1.9 μm) for the A AR and no appreciable affinity for the A and A ARs. Evaluation of their efficacy profiles by measurement of intracellular cAMP levels revealed that 4 and 5 behave as non-nucleoside agonists of the A AR with EC values of 0.47 and 0.87 μm, respectively. No clear concentration-response curves could be instead obtained for 6, probably because this compound modulates one or more additional targets, thus masking the putative effects exerted by its activation of A AR. The three compounds were not able to modulate A AR-mediated cAMP accumulation induced by the non-selective AR agonist NECA, thus demonstrating no affinity toward this receptor.
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http://dx.doi.org/10.1111/cbdd.12801DOI Listing
November 2016

Toward PET imaging of A2B adenosine receptors: a carbon-11 labeled triazinobenzimidazole tracer: Synthesis and imaging of a new A2B PET tracer.

Nucl Med Biol 2016 05 27;43(5):309-17. Epub 2016 Feb 27.

Institute of Clinical Physiology, Consiglio Nazionale delle Ricerche (CNR), Via Moruzzi, 1, I-56124 Pisa, Italy; Fondazione Toscana G. Monasterio, Via Moruzzi, 1, I-56124, Pisa, Italy.

Introduction: A2B adenosine receptors (ARs) are commonly defined as "danger" sensors because they are triggered during cell injury when the endogenous molecule, adenosine, increases rapidly. These receptors, together with the other receptor subtypes (A1, A2A and A3), exert a wide variety of immunomodulating and (cyto)protective effects, thus representing a pivotal therapeutic target for different pathologies including diabetes, tumors, cardiovascular diseases, pulmonary fibrosis and others. The limited availability of potent and selective ligands for A2B ARs has prevented this receptor to emerge both as therapeutic and diagnostic target.

Methods: Recently, a new class of potent A2B ARs antagonists was developed featuring the triazinobenzimidazole scaffold. Starting from this chemotype, we synthesized a new radiotracer, [(11)C]-4 (1-[(11)C]methyl-3-phenyl triazino[4,3-a]benzimidazol-4(1H)-one), and investigated the pharmacokinetics of this compound in vivo to define its potential use in the imaging of A2B AR with positron emission tomography.

Results: [(11)C]-4 showed a very high chemical and blood stability. Results of in vivo and ex vivo experiments underlined the ability of this molecule to bind the A2B AR and correlated with the A2B AR protein and gene expression data.

Conclusions: Although further studies are necessary, these data suggest that [(11)C]-4 may represent a good lead compound for the development of novel selective and potent A2B AR radiotracers, and a new option for the clinical investigation of several pathophysiological processes and chronic diseases.
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http://dx.doi.org/10.1016/j.nucmedbio.2016.02.005DOI Listing
May 2016

A promiscuous recognition mechanism between GPR17 and SDF-1: Molecular insights.

Cell Signal 2016 Jun 10;28(6):631-42. Epub 2016 Mar 10.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy; Dipartimento di Scienze Biomediche e Cliniche "L. Sacco", Università degli Studi di Milano, Via Gian Battista Grassi 74, 20157 Milano, Italy. Electronic address:

Recent data and publications suggest a promiscuous behaviour for GPR17, a class-A GPCR operated by different classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. This observation, together with the ability of several class-A GPCRs to form homo- and hetero-dimers, is likely to unveil new pathophysiological roles and novel emerging pharmacological properties for some of these GPCRs, including GPR17. This receptor shares structural, phylogenetic and functional properties with some chemokine receptors, CXCRs. Both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCR ligands have been demonstrated to have a role in orchestrating inflammatory responses and oligodendrocyte precursor cell differentiation to myelinating cells in acute and chronic diseases of the central nervous system. Here, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary oligodendrocyte precursor cells, we demonstrate that GPR17 can be activated by SDF-1, a ligand of chemokine receptors CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to different classes of GPCR ligands suggests that this receptor modifies its function depending on the extracellular mileu changes occurring under specific pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an inflammatory/immune component.
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http://dx.doi.org/10.1016/j.cellsig.2016.03.001DOI Listing
June 2016

New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line.

Int J Biochem Cell Biol 2016 May 3;74:95-108. Epub 2016 Mar 3.

Department of Pharmacy, University of Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy; Interdepartmental Research Center "Nutraceuticals and Food for Health", University of Pisa, via del Borghetto 80, 56124 Pisa, Italy.

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule.
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http://dx.doi.org/10.1016/j.biocel.2016.02.019DOI Listing
May 2016

Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death.

Sci Rep 2015 Oct 23;5:15556. Epub 2015 Oct 23.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.

Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type.
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http://dx.doi.org/10.1038/srep15556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4616042PMC
October 2015

♦Copper (II) ions modulate Angiogenin activity in human endothelial cells.

Int J Biochem Cell Biol 2015 Mar 14;60:185-96. Epub 2015 Jan 14.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.

Angiogenin (ANG), a member of the secreted ribonuclease family, is a potent angiogenesis stimulator that interacts with endothelial cells inducing a wide range of responses. Metal ions dyshomeostasis play a fundamental role in the onset of neurodegenerative diseases, in particular copper that is also involved in angiogenesis processes. It is known that vascular pathologies are present in neurodegenerative diseases and Angiogenin is down-regulated in Alzheimer and Parkinson diseases, as well as it has been found as one of the mutated genes in amyotrophic lateral sclerosis (ALS). Copper (II) induces an increase of Angiogenin binding to endothelial cells but, so far, the relationship between copper-ANG and angiogenesis induction remain unclear. Herein, the effects of copper (II) ions on Angiogenin activity and expression were evaluated. The binding of copper was demonstrated to affect the intracellular localization of the protein decreasing its nuclear translocation. Moreover, the ANG-copper (II) system negatively affects the protein-induced angiogenesis, as well as endothelial cells migration. Surprisingly, copper also reveals the ability to modulate the Angiogenin transcription. These results highlight the tight relationship between copper and Angiogenin, pointing out the biological relevance of ANG-copper system in the regulation of endothelial cell function, and revealing a possible new mechanism at the basis of vascular pathologies.
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http://dx.doi.org/10.1016/j.biocel.2015.01.005DOI Listing
March 2015

Further studies on pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as potent and selective human A1 adenosine receptor antagonists.

Eur J Med Chem 2015 Jan 12;89:32-41. Epub 2014 Oct 12.

Dipartimento NEUROFARBA, Sezione Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy.

A new series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was synthesized and tested in radioligand binding assays on human A1, A2A and A3 adenosine receptors. Most of the compounds showed high selectivity of action towards A1 receptor and high affinity with Ki values in the low nanomolar range. The pharmacological profile of the most active molecules towards A1 adenosine receptors was evaluated in cAMP functional assay. Compounds demonstrated their ability to completely counteract the effect of the agonist NECA, thus demonstrating their antagonist profile. Moreover, the most interesting compound, tested in the mouse passive avoidance, exhibited an antiamnesic effect at the doses of 10 and 30 mg/kg.
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http://dx.doi.org/10.1016/j.ejmech.2014.10.020DOI Listing
January 2015

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators.

Biochim Biophys Acta 2014 Dec 18;1843(12):2957-66. Epub 2014 Sep 18.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. Electronic address:

The A2B adenosine receptor (A2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases. Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization. In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A2B AR in the control of osteoblast differentiation, bone formation and fracture repair.
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http://dx.doi.org/10.1016/j.bbamcr.2014.09.013DOI Listing
December 2014