Design and synthesis of novel compounds interacting with modulatory cysteines of MGL

N-arachidonoyletanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are the most representative members of a class of endogenous ligands named endocannabinoid, which are able to activate cannabinoids receptors. The endocannabinoid neuromodulation plays important roles in various aspects of neural functions including learning and memory, anxiety, depression, appetite and feeding behavior, nociception, neuroprotection and movement control. Since 2-AG has been considered the true on-demand released endocannabinoid ligand for CB1 receptor, a promising way to explore and promote its therapeutic potential could be the selective inhibition of the monoacylglycerol lipase (MGL), the enzyme responsible of 2-AG degradation. Three critical residues of cysteine have been identified as regulatory gates for the activity of the enzyme: Cys242, within the active site close to the catalytic triad, Cys201 and Cys208 located on the lid domain, a flexible solvent-exposed region that regulate the access of the substrate to the active site. Cys201 and Cys208 have been recently shown to be redox switches that regulate the activity of MGL by undergoing oxidation after oxidative stimulus induced by hydrogen peroxide. Despite the potential of MGL as target for 2-AG signaling modulation, few potent and selective MGL inhibitors are available until now. Recently two class of partially-reversible MGL inhibitors has been reported: one is populated by covalent disulfide-bond forming compounds such as isothiazol-3(2H)-one (ITZ) and benzo[d]isothiazol-3(2H)-one (BTZ) derivatives, the second one is populated by triterpenes such as euphol and pristimerin. Both these classes of compounds interact with critical residues Cys201 and Cys208 of MGL thus preserving the catalytic activity of the enzyme. The modulation of MGL activity by interaction with these critical cysteine residues is a crucial point to fine tune the 2-AG signaling avoiding behavioral side-effects associated to the total disruption of the catalytic activity of MGL exerted by inhibitors addressing the catalytic site The research work reported in this PhD dissertation has been focused on the exploration of the structure-activity relationships (SAR) around the BTZ warhead in order to explore the pharmacophoric space around the targeted cysteines of MGL. The structure of the driver portion of compounds sharing an identical warhead has been broadly changed. I investigated the effect of substituent groups with different steric demand and electronic inductive effects on the nitrogen atom and the substituents on the phenyl ring of BTZ. I performed the design and the synthesis of several compounds, applying different synthetic strategies. The inhibitory potency of synthesized BTZs on MGL and was evaluated and SAR of this class were described. We identified promising compounds with inhibitory potency on MGL in the low nanomolar range. Moreover, the lead compound emerged from this screening was subjected to mechanistic studies that revealed a behavior similar to that of hydrogen peroxide in inactivating MGL supporting the theory that BTZs mimic the effect of an endogenous oxidation stimulus. To further elucidate the role of critical Cys201 and Cys208 in MGL activity, site-directed mutagenesis studies were performed identifying both cysteines as possible counterpart in the interaction with BTZs. Moreover, in order to evaluate the stability and reactivity of these compounds towards biological thiols we investigated, trough analytical methods the effect of substituent groups with different steric hindrance and electronic effects on the sulfenamidic reactive core of BTZ. I investigated by means of NMR experiments the reactivity of selected BTZs with a model biological thiol, N-acetyl cysteine (NAC). These studies allowed us to define a structure-reactivity relationship landscape that further validated our biological results. To increse the knolewdge in the field of the terpene-based inhibitors of MGL, I synthesized 3 new compounds starting form commercially available celastrol: these scaffold modifications led to less active compounds than reference compound pristimerin thus discouraging the development of triperpene-based MGL inhibitors. Fibroblast growth factor-2 (FGF2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF2 exerts its activity on endothelial cells by interacting with high affinity tyrosine-kinase FGF receptors (FGFRs) and low affinity heparan sulphate proteoglycans (HSPGs), leading to the formation of productive HSPG/FGF2/FGFR ternary complexes. Presta and coworkers identified, from a virtual screening study of an NCI library, a promising molecule NSC172288 able to inhibit FGF2-FGFR interactions. During my PhD research I designed and performed the synthesis of this compound not described in literature. The major challenge associated was the necessity of planning the synthesis on the base of a poorly described structure. A synthetic pathway for NSC172285 was setup and an efficient method for using HFA trihydrate as electrophilic partner in an aldol reaction, with standard laboratory equipment was developed

Synthesis, Structural Elucidation, and Biological Evaluation of NSC12, an Orally Available Fibroblast Growth Factor (FGF) Ligand Trap for the Treatment of FGF-Dependent Lung Tumors

NSC12 is an orally available pan-FGF trap able to inhibit FGF2/FGFR interaction and endowed with promising antitumor activity. It was identified by virtual screening from a NCI small molecule library, but no data were available about its synthesis, stereochemistry, and physicochemical properties. We report here a synthetic route that allowed us to characterize and unambiguously identify the structure of the active compound by a combination of NMR spectroscopy and in silico conformational analysis. The synthetic protocol allowed us to sustain experiments aimed at assessing its therapeutic potential for the treatment of FGF-dependent lung cancers. A crucial step in the synthesis generated a couple of diastereoisomers, with only one able to act as a FGF trap molecule and to inhibit FGF-dependent receptor activation, cell proliferation, and tumor growth when tested in vitro and in vivo on murine and human lung cancer cells

Potential Use of Tea Tree Oil as a Disinfectant Agent against Coronaviruses: A Combined Experimental and Simulation Study

: The COVID-19 pandemic has highlighted the relevance of proper disinfection procedures and renewed interest in developing novel disinfectant materials as a preventive strategy to limit SARS-CoV-2 contamination. Given its widely known antibacterial, antifungal, and antiviral properties, Melaleuca alternifolia essential oil, also named Tea tree oil (TTO), is recognized as a potential effective and safe natural disinfectant agent. In particular, the proposed antiviral activity of TTO involves the inhibition of viral entry and fusion, interfering with the structural dynamics of the membrane and with the protein envelope components. In this study, for the first time, we demonstrated the virucidal effects of TTO against the feline coronavirus (FCoVII) and the human coronavirus OC43 (HCoV-OC43), both used as surrogate models for SARS-CoV-2. Then, to atomistically uncover the possible effects exerted by TTO compounds on the outer surface of the SARS-CoV-2 virion, we performed Gaussian accelerated Molecular Dynamics simulations of a SARS-CoV-2 envelope portion, including a complete model of the Spike glycoprotein in the absence or presence of the three main TTO compounds (terpinen-4-ol, γ-terpinene, and 1,8-cineole). The obtained results allowed us to hypothesize the mechanism of action of TTO and its possible use as an anti-coronavirus disinfectant agent

Hydrophilic 1,10-phenanthroline derivatives for selective Am(III) stripping into aqueous solutions

The novel and fully combustible hydrophilic 1,10-phenanthroline-2,9-dicarboxamide (1) was synthesized and investigated as Am(III) stripping agent in a simulated advanced hydrometallurgical process, in comparison with two other 1,10-phenanthroline-based ligands 2 and 3. The stripping efficiency and the Am(III)/lanthanides(III) selectivity of the TODGA (org)/phen-derivative (aq) extracting system were studied under several experimental conditions by liquid–liquid extraction tests. The results obtained clarify the main limitations of these ligands in the scope of the hydrometallurgical reprocessing but also enable to get indications to steer future investigations in the domain of the selective An(III) recovery for the advanced reprocessing of Spent Nuclear Fuel by hydrophilic ligands

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors

How do cardiologists select patients for dual antiplatelet therapy continuation beyond 1 year after a myocardial infarction? Insights from the EYESHOT Post-MI Study

Background: Current guidelines suggest to consider dual antiplatelet therapy (DAPT) continuation for longer than 12 months in selected patients with myocardial infarction (MI). Hypothesis: We sought to assess the criteria used by cardiologists in daily practice to select patients with a history of MI eligible for DAPT continuation beyond 1 year. Methods: We analyzed data from the EYESHOT Post-MI, a prospective, observational, nationwide study aimed to evaluate the management of patients presenting to cardiologists 1 to 3 years from the last MI event. Results: Out of the 1633 post-MI patients enrolled in the study between March and December 2017, 557 (34.1%) were on DAPT at the time of enrolment, and 450 (27.6%) were prescribed DAPT after cardiologist assessment. At multivariate analyses, a percutaneous coronary intervention (PCI) with multiple stents and the presence of peripheral artery disease (PAD) resulted as independent predictors of DAPT continuation, while atrial fibrillation was the only independent predictor of DAPT interruption for patients both at the second and the third year from MI at enrolment and the time of discharge/end of the visit. Conclusions: Risk scores recommended by current guidelines for guiding decisions on DAPT duration are underused and misused in clinical practice. A PCI with multiple stents and a history of PAD resulted as the clinical variables more frequently associated with DAPT continuation beyond 1 year from the index MI

EVALITA Evaluation of NLP and Speech Tools for Italian - December 17th, 2020

Welcome to EVALITA 2020! EVALITA is the evaluation campaign of Natural Language Processing and Speech Tools for Italian. EVALITA is an initiative of the Italian Association for Computational Linguistics (AILC, http://www.ai-lc.it) and it is endorsed by the Italian Association for Artificial Intelligence (AIxIA, http://www.aixia.it) and the Italian Association for Speech Sciences (AISV, http://www.aisv.it)

Giacomo Balla, Portrait of Fontana, 1907 (Ritratto di Fontana)

Analisi del dipinto 'Ritratto di Fontana' di Giacomo Balla, di proprietà della Estorick Collection di Londra. Un attento esame dell'opera, basato anche su indagini multispettrali, sulla radiografia X e sulla riflettografia multispettrale a scanner, ha permesso di chiarire che il dipinto non può essere identificato come ritratto dello scultore Carlo Fontana, come si credeva. Le indagini scientifiche hanno permesso di individuare numerosi aspetti tecnici caratteristici della produzione prefuturista di Balla

Umberto Boccioni, Portrait of Betty and Nora Baer, 1909 (Ritratto di Betty e Nora Baer)

Presentazione delle ricerche e dei risultati delle indagini scientifiche sul dipinto 'Ritratto di Betty e Nora Baer', opera di collezione privata presente nel catalogo ragionato di Umberto Boccioni ma mai, prima d'ora, indagata approfonditamente. Il contributo fa luce sulla vicenda collezionistica e sulla tecnica pittorica adottata dall'artista

Giacomo Balla, The Hand of the Violinist, 1912 (La mano del violinista / Ritmi dell'archetto)

Presentazione delle indagini multidisciplinari effettuate su 'La mano del violinista' (noto anche come 'Ritmi dell'archetto'), opera cruciale nel percorso di Giacomo Balla. Le ricerche archivistico-bibliografiche, unitamente alle indagini scientifiche, hanno permesso di ottenere risultati rilevanti per la conoscenza di questo dipinto. Si è per la prima volta chiarita la complessa storia collezionistica dell'opera, sciogliendo nodi ed equivoci che avevano confuso gli studiosi dell'artista; è stato scoperto un dipinto soggiacente la superficie pittorica e riferibile al ciclo decorativo che Balla realizzò per la casa dei coniugi Lowenstein a Dusseldorf tra l'estate e l'ottobre del 1912; sono stati messi a fuoco numerosi aspetti legati alla realizzazione dell'opera e al ruolo della cornice, costruita in perfetta armonia con il progetto compositivo dell'opera