Selectively Charged and Zwitterionic Analogues of the Smallest Immunogenic Structure of Streptococcus Pneumoniae Type 14

Zwitterionic polysaccharides (ZPs) have been shown in recent years to display peculiar immunological properties, thus attracting the interest of the carbohydrate research community. To fully elucidate the mechanisms underlying these properties and exploit the potential of this kind of structures, in depth studies are still required. In this context, the preparation of two cationic, an anionic, as well as two zwitterionic tetrasaccharide analogues of the smallest immunogenic structure of Streptococcus pneumoniae type 14 (SP14) capsular polysaccharide are presented. By exploiting a block strategy, the negative charge has been installed on the non-reducing end of the lactose unit of the tetrasaccharide and the positive charge either on the non-reducing end of the lactosamine moiety or on an external linker. These structures have then been tested by competitive ELISA, showing that the structural variations we made do not modify the affnity of the neutral compound to binding to a specific antibody. However, lower effcacies than the natural SP14 compound were observed. The results obtained, although promising, point to the need to further elongate the polysaccharide structure, which is likely too short to cover the entire epitopes

Synthesis and Evaluation of Monoaryl Derivatives as Transthyretin Fibril Formation Inhibitors

Transthyretin (TTR) is a homo-tetrameric protein characterized by four identical β-sheet rich monomers assembled together to form a tetramer that is crossed, along the 2-fold symmetry axis, by two similar binding pockets named thyroxin binding sites. Under unknown conditions, TTR can misfold and aggregate triggering the amyloidosis onset. One therapeutic approach consists to stabilize the tetramer with synthetic small molecules that bind TTR binding site hindering the first step of fibril formation. Here, we report the synthesis of new 2-((benzyloxy)imino)acetic, -propanoic and -butanoic acid derivatives, results of their turbidimetric UV assay and the docking study of new monoaryl compounds. The obtained results suggest that, for this class of compounds, (i) the chlorine atom in ortho position on the aromatic ring is the best substituent; (ii) the linker inversion still allows the interaction with thyroxine binding sites; and (iii) the steric hindrance in R1 position is detrimental for the activity

Ionic liquid-promoted green synthesis of biologically relevant diaryl thioethers

This study reports an ionic liquid (IL) promoted green method to obtain diaryl thioethers useful as key intermediates for the synthesis of matrix metalloproteinase (MMP) inhibitors. The synthetic pathway includes a sequential Ullmann reaction and Suzuki cross-coupling. The Ullmann conditions were optimized as regards the catalyst, the ionic liquid, reaction time, and temperature. Under optimal conditions (1-butyl-3-methylimidazolium bromide ([BMIM]Br) as solvent; catalyst, Cu; base, K2CO3; reaction time, 22 h; reaction temperature, 150°C) the aryl iodide conversion was 95%. The Suzuki cross-coupling was conducted in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4) at 110°C, using Pd(PPh3)4 as catalyst and Na2CO3 as base. For both reactions, a combination of ILs with microwave (MW) irradiation determined a substantial improvement of reaction time and yields compared to conventional heating. This multi-step process reduces reaction times and removes organic solvents providing a more eco-friendly alternative for the synthesis of important pharmaceutical building blocks

Discovery of Dimeric Arylsulfonamides as Potent ADAM8 Inhibitors

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration and angiogenesis. The use of bivalent inhibitors could impair migration and invasion, through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and MMPs. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 showed to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays, at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8

The metalloproteinase ADAM10 requires its activity to sustain surface expression

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss

Identification of a Novel p53 Modulator Endowed with Antitumoural and Antibacterial Activity through a Scaffold Repurposing Approach

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection

Design, Synthesis and Development of Hit Compounds able to Modulate some Bio-Pharmacological Activities of Metalloenzymes Involved in Degenerative Diseases

Matrix Metalloproteinases (MMPs) are a family of enzymes that are attracting growing interest as therapeutic targets. They are proteases whose fundamental role has been recognised in the degradation of the Extracellular Matrix (ECM) components. Their expression is finely regulated at many levels (transcription, activation, inhibition) while unregulated profiles have been found in many pathological conditions such as cancer, arthritis, atherosclerosis, and inflammatory diseases. Since the use of endogenous inhibitors is not easy to accomplish, the design and synthesis of new small molecules that could allow the regulation of these proteins is an avanguard in nowadays research of new anticancer drugs, as well as for other therapies. The research project of my PhD thesis focused mainly on four topics: Synthesis and biological evaluation of arylsulfones and thioaryl derivatives bearing alternative zinc-binding groups as MMP-12 inhibitors Synthesis and biological evaluation of new sugar-based arylsulfonamide carboxylates as MMP- 12 inhibitors with improved water solubility Synthesis and biological evaluation of bifunctional ligands of MT1-MMP Biological evaluation of selective ADAM-10 inhibitors able to inhibit DDR1 and DDR2 sheddin

Sintesi di eterodimeri contenenti unità di N-acetil-D-glucosamina come potenziali inibitori di metalloproteinasi (MMPs)

Le metalloproteinasi della matrice (MMPs), dette anche matrixine, sono una famiglia di enzimi metallodipendenti normalmante presenti nella matrice extracellulare e rappresentano un gruppo di endopeptidasi multidominio zinco dipendenti ad attività proteolitica nei confronti di proteine della matrice extracellulare (ECM) e di varie proteine non-ECM. Le MMPs svolgono una funzione centrale nei processi fisiologici di accrescimento, sviluppo e rimodellamento tissutale. In condizioni non patologiche la regolazione, da parte di specifici inibitori tissutali delle MMPs, esercita un ruolo chiave per mantenere l’equilibro tra i processi di distruzione e generazione dei costituenti della matrice extracellulare [1]. La compromissione di questo equilibrio a causa di una iperattivazione delle MMPs, oltre a determinare l’insorgenza di numerose patologie degenerative e infiammatorie (artrite reumatoide, osteoartrite e psoriasi), malattie cardiovascolari e neurodegenerative, svolge un ruolo primario nell’attivazione linfocitaria e nella progressione tumorale. Ad esempio, mentre le MMP-12 e le MMP-9 sono responsabili della broncopneumopatia ostruttiva cronica (COPD), le MMP-8 sono anche iper-espresse in varie neoplasie e la loro iper-espressione è correlata all’aggressività tumorale e al potenziale metastatico. Ne consegue che la sintesi di nuovi inibitori di MMPs ad alta selettività per il controllo dell’attività proteolitica di questi enzimi sia tutt’oggi molto interessante, sia dal punto di vista farmacologico che diagnostico. Analizzando il sito di binding, in generale, sono stati definite due porzioni fondamentali per l’attività di ipotetici inibitori: il sito L, necessario per chelare lo ione zinco presente nel sito attivo dell’enzima, nel quale si osserva una buona interazione con gruppi funzionali come un idrossammato o un carbossilato e la porzione S1’ ad alta affinità nei confronti di subunità lipofile e planari. Le N-acetil-D-esosammine, amminozuccheri dove il gruppo ossidrilico in posizione 2 è sostituito con un gruppo N-acetilammidico, sono tra i carboidrati più rappresentati nelle macromolecole saccaridiche come, ad esempio, le glicoproteine e i glicolipidi. Questi saccaridi, inseriti in strutture più complesse, sono responsabili di numerosi processi biologici, che vanno dal riconoscimento ed adesione tra cellule fino alla difesa immunitaria e la replicazione virale. Composti di tale struttura sono, quindi, strettamente correlati con lo sviluppo di processi infiammatori, quali l’osteoartrite e l’artrite reumatoide, infezioni virali e batteriche [2]. Le N-acetil-D-esosammine più importanti e diffuse in natura, ottenibili dall’idrolisi acida di macromolecole di origine naturale, sono la N-acetil-D-galattosammina (GalNAc), la N-acetil-D-glucosammina (GlcNAc) e la N-acetil-D-mannosammina (ManNAc). La ricerca svolta in questa tesi, ha avuto come scopo quello di coniugare strutture solfonammidiche, con nota attività di inibizione nei confronti delle MMPs, con un’unità di N-acetil-D-glucosammina a formare eterodimeri in grado di esplicare una più selettiva attività inibitoria verso le MMP-8, MMP-9 e MMP-12. Per realizzare il coupling delle suddette unità sono state scartate tecniche di coniugazione che comprendessero la formazione di legami ammidici e esterei, in quanto labili in presenza dei comuni enzimi presenti in vivo come esterasi e amidasi. Sono stati prediletti i coupling attraverso la formazione sia di un legame tiureidico e sia di un legame 1,2,3-triazolico, facilmente ottenibile attraverso una cicloaddizione 1,3-dipolare, nota reazione di “click chemistry”. Mentre il legame tioureidico mostra una buona stabilità e, ipoteticamente, potrebbe essere fonte di ulteriori interazioni deboli con il sito di binding, studi recenti hanno evidenziato che il nucleo l’1,2,3-triazolico incrementa l’attività inibitoria nei confronti delle MMPs [3]. La preparazione degli eterodimeri ha previsto la preliminare preparazione di: 1) idonei precursori N-acetilglucosamminici portanti un gruppo isotiocianato in grado di generare il legame tioureidico con l’unità solfonammidica avente uno spaziatore funzionalizzato con un gruppo amminico. 2) idonei precursori N-acetilglucosamminici portanti un gruppo azidico in grado di generare il legame 1,2,3-triazolico con l’unità solfonammidica avente uno spaziatore funzionalizzato con un gruppo alchinico terminale. Con questa strategia sono stati preparati eterodimeri aventi una distanza variabile tra la porzione zuccherina e l’inibitore di MMPs con lo scopo di valutarne l’attività inibitoria verso le MMPs in dipendenza dal tipo e dalla lunghezza del linker. Esperimenti NMR mono (1H, 13C) e bidimensionali (COSY, HSQC, DEPT-135) hanno permesso di determinare la struttura e la stereochimica di tutti i prodotti ottenuti in questa Tesi ed i dati spettroscopici sono concordi con le strutture proposte. Gli eterodimeri opportunamente deprotetti, ottenuti in questa tesi, sono stati saggiati in vitro sugli enzimi umani ricombinanti MMP-8, MMP-9 e MMP-12 e hanno evidenziato un’attività inibitoria soddisfacente dell’ordine nanomolare. Le informazioni che saranno ricavate dall’analisi strutturale dei complessi cristallini enzima-eterodimero inibitore, attraverso cristallografia a raggi X, saranno estremamente importanti nella progettazione e sintesi di nuovi potenziali e selettivi inibitori delle MMPs. Bibliografia 1Murphy, G.; Nagase, H.; Progress in matrix metalloproteinase research, Molecular Aspects of Medicine 2008, 29, 290-308 2 a) Horton, D.; Wander, J. D.; The Carbohydrates: Chemistry and Biochemistry, 2nd. Ed; b)El Sayed, H.E.A.; Mohamed, R.E.A.; Pure Appl. Chem. 2007, 79, 2229-2242 3 (a) Hugenberg, V.; Riemann, B.; Hermann, S.; Schober, O.; Schäfers, M.; Szardenings, K.; Lebedev, A.; Gangadharmath, U.; Kolb, H.; Walsh, J.; Zhang, W.; Kopka, K.; Wagner, S. J. Med. Chem. 2013, 56, 6858-6870; (b) Hugenberg, V.; Breyholz, H-J.;Riemann, B.; Hermann, S.; Schober, O.; Schäfers, M.; Gangadharmath, U.; Mocharla, V.; Kolb, H.; Walsh, J.; Zhang, W.; Kopka, K.; Wagner, S. J. Med. Chem. 2012, 55, 4714-4727

Dietary Bioactive Compounds: Implications for Oxidative Stress and Inflammation

Nowadays, it has been amply demonstrated how an appropriate diet and lifestyle are essential for preserving wellbeing and preventing illnesses [...

An overview of carbohydrate-based carbonic anhydrase inhibitors

Carbonic anhydrases (CAs) are metalloenzymes responsible for the reversible hydration of carbon dioxide to bicarbonate, a fundamental reaction involved in various physiological and pathological processes. In the last decades, CAs have been considered as important drug targets for different pathologies such as glaucoma, epilepsy and cancer. The design of potent and selective inhibitors has been an outstanding goal leading to the discovery of new drugs. Among the different strategies developed to date, the design of carbohydrate-based CA inhibitors (CAIs) has emerged as a versatile tool in order to selectively target CAs. The insertion of a glycosyl moiety as a hydrophilic tail in sulfonamide, sulfenamide, sulfamate or coumarin scaffolds allowed the discovery of many different series of sugar-based CAIs, with relevant inhibitory results. This review will focus on carbohydrate-based CAIs developed so far, classifying them in glycosidic and glycoconjugated inhibitors based on the conjugation chemistry adopted