“Gli aspetti innovativi dell’informativa finanziaria nelle “entità” del settore pubblico: gli IPSAS 5, 15, 18, 19 e 21”

Per il citato lavoro, che rappresenta un contributo alla ricerca della Scuola Superiore della Pubblica Amministrazione “Principi e metodi di contabilità economico-patrimoniale per lo Stato e le Pubbliche Amministrazioni nel quadro teorico ed operativo internazionale” (coordinatore Prof. L. Anselmi, anni 2007/2008), sono stati assolti gli obblighi di pubblicazione direttamente dall’autore, ai sensi della Legge 15.4.2006 n. 106 e del D.P.R. 03.05.2006, n. 252

Développement d'inhibiteurs d'endonucléases de Bunyavirales par chimie click in situ

Les virus de l'ordre des Bunyavirales constituent un problème de santé publique en raison du manque d’options thérapeutiques et de leur répartition mondiale. La protéine L gouvernant le système de réplication virale possède une activité endonucléase responsable du mécanisme du vol-de-coiffe permettant la transcription. Cette enzyme clé a été identifiée comme une cible thérapeutique de choix pour développer des antiviraux. Son mécanisme d’hydrolyse de l’ARN médié par les ions Mg2+ permet de développer des inhibiteurs chélateurs de métaux de type dicéto-acide (DCA). La synthèse guidée par la cible biologique est une méthode puissante qui met au centre la cible thérapeutique qui assemble ses propres inhibiteurs en son site-actif. Nous décrivons l’utilisation des endonucléases de Bunyavirales comme « moule de réaction » pour la génération de leurs propres inhibiteurs chélateurs de métaux. Une librairie de 25 composés N-triazolyl-DCA (NT-DCA), C-triazolyl-DCA (CT-DCA) et C-triazolyl-DHP (CT-DHP) a été générée par des voies de synthèses nouvelles par chimie click. Les composés ont été caractérisés biophysiquement par Thermophorèse à Micro-échelle (MST) et par Fluorimétrie Différentielle à Balayage (DSF) et leur pouvoir inhibiteur a été évalué in vitro et dans des cellules infectées. De plus, la synthèse d’un ligand fluorescéine-DCA a été conduite pour développer un test par compétition par Polarisation de Fluorescence (FP) pour tester l’activité des composés. Des ligands à haute affinité possédant une forte activité in vitro et en cellules infectées ont été identifiés. Dans le contexte de la pandémie de COVID-19, la librairie a été repositionnée sur l'exonucléase du SARS-CoV-2.With a worldwide repartition and limited therapeutic options Bunyavirales viruses represent a major public health issue. The replication machinery of these viruses is governed by the intricate L-protein that displays an endonuclease activity responsible for the cap-snatching mechanism that allows viral transcription. This key enzyme was identified as a promising target to develop antivirals. Its catalytic mechanism of RNA hydrolysis mediated by Mg2+ ions enables the development of diketo acid (DKA) metal-chelating inhibitors. Target-Guided-Synthesis (TGS) is a powerful method that directly involves the therapeutic target that assembles its own inhibitors in its active site like LEGOs®. Herein, we describe the use of Bunyavirales endonucleases as reaction vessels for the In Situ generation of metal-chelating inhibitors. A library of 25 N-triazolyl-DKA (NT-DKA), C-triazolyl-DKA (CT-DKA) and C-triazolyl-DHP (CT-DHP) compounds was generated with new and original synthesis pathways via click chemistry. The compounds were biophysically characterized by Microscale Thermophoresis (MST) and Differential Scanning Fluorimetry (DSF) and their inhibitory potency was evaluated in vitro and in infected cells culture. Moreover, a fluorescein-DKA ligand was synthesized for the development of a competition Fluorescence Polarization (FP) assay to determine the activity of the compounds. High-affinity ligands that demonstrated a potent efficiency in vitro and in cellula were identified. In the context of the COVID-19 pandemic, the compounds library was repositioned against SARS-CoV-2 exonuclease which opened new possibilities for the development of metal-chelating Coronavirus inhibitors

Efficient access to 3′-deoxy-3′-(4-substituted-1,2,3-triazol-1-yl)-thymidine derivatives via ligand-promoted CuAAC

International audienceHerein we describe an efficient and rapid access to 3 0-deoxy-3 0-(4-substituted-1,2,3-triazol-1-yl)thymidine derivatives using 1,3-dipolar cycloaddition reaction catalyzed by copper(I). Innovative conditions allow us to generate target compounds in a one-pot reaction mixing 3 0-azido-3 0-deoxythymidine, alkyne, copper sulfate pentahydrate, sodium ascorbate and tris(benzyltriazolylmethyl)amine in a water:tert-butanol solvent mixture. Rapid treatment of the reaction and subsequent flash purification chromatography afforded pure compounds in an overall yield of 71e100%. All eleven synthesized compounds were identified on the basis of their spectral data analysis (1 H, 13 C, 2D NMR and highresolution mass spectra)

A high-throughput fluorescence polarization assay to discover inhibitors of arenavirus and coronavirus exoribonucleases

Abstract Viral exoribonucleases are uncommon in the world of RNA viruses. To date, this activity has been identified only in the Arenaviridae and the Coronaviridae families. These exoribonucleases play important but different roles in both families: for mammarenaviruses the exoribonuclease is involved in the suppression of the host immune response whereas for coronaviruses, exoribonuclease is both involved in a proofreading mechanism ensuring the genetic stability of viral genomes and participating to evasion of the host innate immunity. Because of their key roles, they constitute attractive targets for drug development. Here we present a high-throughput assay using fluorescence polarization to assess the viral exoribonuclease activity and its inhibition. We validate the assay using three different viral enzymes from SARS-CoV-2, lymphocytic choriomeningitis and Machupo viruses. The method is sensitive, robust, amenable to miniaturization (384 well plates) and allowed us to validate the proof-of-concept of the assay by screening a small focused compounds library (23 metal chelators). We also determined the IC50 of one inhibitor common to the three viruses. Highlights Arenaviridae and Coronaviridae viral families share an exoribonuclease activity of common evolutionary origin Arenaviridae and Coronaviridae exoribonuclease is an attractive target for drug development We present a high-throughput assay in 384 well-plates for the screening of inhibitors using fluorescence polarization We validated the assay by screening of a focused library of 23 metal chelators against SARS-CoV-2, Lymphocytic Choriomeningitis virus and Machupo virus exoribonucleases We determined the IC 50 by fluorescence polarization of one inhibitor common to the three viruses

Structural characterisation and inhibition of Arenavirus replication complex elements : assembly, function and inhibition of embedded nucleases

Arenaviruses, belongs to a family of emerging enveloped segmented and ambisens RNA viruses associated withneurological and hemorrhagic diseases in humans. Arenavirus transcription and genome replication are cytoplasmic ensured by aribonucleoproteine replicase complex NP-L. After penetration, L protein initiates transcription to produce NP and L mRNAs[ 1].The priming of transcription is the result of a cap-snatching mechanism ensured by an endonuclease domain associated to the Lpolymerase. As the concentration of NP in the cell increases, genome segments are replicated, to produce full-length copies(cRNA). cRNAs are now templates for transcription of GPC mRNA (from the S segment) and Z mRNA (from the L segment).The NP caries an exonuclease in charge of clearing out from the cytoplasm dsRNA triggering innate immunity response. Bothnucleases have a similar two metal ion catalytic mechanism, with the particularity of transitioning ion brought by the RNAsubstrate. Any alteration of the remaining ion impairs greatly theses activities[2]. We present a global study aiming to characterizethe assembly of the NP[3], through flexible domains[4], a step critical for vRNApackaging and the polsitioning of L for vRNAreplication, as well as using a combined approach of biophysical screening, crystallography and in silico docking, identifyingactive compounds against both nucleases[5]. Crystal structures of the nucleases domain complexed with several compounds wereobtained[67]. By developing specific compounds to alter both transcription and innate immunity shadowing, our strategy is togive the cell a fighting chance to clear the infection. Combining structure, enzymology, rational synthesis, hit-To-leadoptimization, in cellula evaluation, and screening methods, we are presenting the results of a 2nd generation of molecules pavingthe way to the design of a 3rd generation increasing specificity towards Arenaviral nucleases in the context of the replicationcomplex[8]

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay

International audienceViral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design.Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells

Pomegranate Extract Affects Gut Biofilm Forming Bacteria and Promotes Intestinal Mucosal Healing Regulating the Crosstalk between Epithelial Cells and Intestinal Fibroblasts

Background: Pomegranate (Punica granatum) can be used to prepare a bioactive extract exerting anti-inflammatory activities. Clinical studies demonstrated an improvement in clinical response in inflammatory bowel disease (IBD) patients when pomegranate extract (PG) was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. This study investigates the effect of PG on bacterial biofilm formation and the promotion of mucosal wound healing. Methods: The acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) by oral gavage for 11 days. Colitis was scored daily by evaluating body weight loss, bleeding, and stool consistency. In vivo intestinal permeability was evaluated by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation was assessed by fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity were monitored by immunostaining for JAM-A and MUC-2 markers. Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 h. Primary fibroblasts were isolated from healthy and inflamed areas of 8 IBD patients, and Caco-2 cells were stimulated with or without PG (5 μg/mL). Inflammatory mediators were measured at the mRNA and protein level by RT-PCR, WB, or Bio-plex multiplex immunoassay, respectively. Results: In vivo, PG boosted the recovery phase of colitis, promoting a complete restoration of the intestinal barrier with the regeneration of the mucus layer, as also demonstrated by the absence of bacterial spread into the mucosa and the enrichment of crypt-associated fibroblasts. Microfluidic experiments did not highlight a specific effect of PG on Enterobacterales biofilm formation, even though Citrobacter freundii biofilm was slightly impaired in the presence of PG. In vitro, inflamed fibroblasts responded to PG by downregulating the release of metalloproteinases, IL-6, and IL-8 and upregulating the levels of HGF. Caco-2 cells cultured in a medium supplemented with PG increased the expression of SOX-9 and CD44, whereas in the presence of HGF or plated with a fibroblast-conditioned medium, they displayed a decrease in SOX-9 and CD44 expression and an increase in AXIN2, a negative regulator of Wnt signaling. Conclusions: These data provide new insight into the manifold effects of PG on promoting mucosal homeostasis in IBD by affecting pathogen biofilm formation and favoring the regeneration of the intestinal barrier through the regulation of the crosstalk between epithelial and stromal cells