Sintesi di nuovi inibitori della formazione delle fibrille amiloidi della TTR

La dissociazione di una proteina in monomeri e la sua successiva aggregazione sono in relazione con diverse patologie umane; tra queste l’Alzheimer, il Parkinson, e le amiloidosi, tutte malattie con meccanismi che non sono ancora completamente conosciuti. La Transtiretina (TTR) è una proteina plasmatica deputata principalmente al trasporto della tiroxina e del retinolo; viene sintetizzata nel fegato e nel Sistema Nervoso Centrale (SNC), a livello dei plessi corioidei. Forme di TTR mutata (wild-type), in seguito a denaturazione, sono associate alla sintesi di monomeri alterati, che possono portare alla formazione di fibrille amiloidi, aggregati proteici che, non essendo solubili nei liquidi biologici, si depositano nei tessuti e nelle matrici extracellulari, causando neurodegenerazione e morte cellulare [1]. La deposizione di tali aggregati è alla base dell’insorgenza delle malattie amiloidi. Sono state osservate quattro tipi di amiloidosi: amiloidosi sistemica senile (SSA); cardiomiopatia amiloide familiare (FAC); polineuropatia amiloide familiare (FAP); amiloidosi del sistema nervoso centrale (CNSA). Attualmente l’unico approccio clinico consentito per il trattamento delle patologie legate alla TTR è il trapianto di fegato, allo scopo di bloccare la sintesi di TTR mutata; alcune mutazioni, tuttavia, possono presentarsi comunque. D’altra parte, non esistono terapie farmacologiche efficaci per combattere la deposizione di TTR wild-type nella SSA. Studi sulla TTR e sul suo ligando endogeno, la Tiroxina, hanno mostrato come T4 sia in grado di stabilizzare lo stato nativo della proteina impedendone la denaturazione e la successiva riaggregazione in fibrille. La struttura della Tiroxina e la sua interazione con il sito di legame della TTR sono stati utilizzati come punto di partenza per studi condotti sui farmaci anti-infiammatori non steroidei (NSAIDs) che hanno mostrato caratteristiche comuni all’ormone endogeno. Il progetto di tesi ha come scopo la sintesi di nuove molecole in grado di stabilizzare la struttura tetramerica della TTR inibendone la denaturazione e la successiva formazione di fibrille. I composti sintetizzati presentano tutti una struttura generale comune nella quale il gruppo carbossilico, importante per l’interazione con il sito di legame del tetramero, non è legato direttamente ad un anello aromatico come è riportato in inibitori noti in letteratura, ma è legato ad una porzione aromatica biciclica o triciclica, variamente sostituita, mediante un linker etero aromatico (C-O-N) flessibile, che ne permette un miglior orientamento nel contesto del sito di legame della proteina

Transthyretin complexes with curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent mixtures

Crystallographic structure determination of protein–ligand complexes of transthyretin (TTR) has been hindered by the low affinity of many compounds that bind to the central cavity of the tetramer. Because crystallization trials are carried out at protein and ligand concentration that approach the millimolar range, low affinity is less of a problem than the poor solubility of many compounds that have been shown to inhibit amyloid fibril formation. To achieve complete occupancy in co-crystallization experiments, the minimal requirement is one ligand for each of the two sites within the TTR tetramer. Here we present a new strategy for the co-crystallization of TTR using high molecular weight polyethylene glycol instead of high ionic strength precipitants, with ligands solubilized in multicomponent mixtures of compounds. This strategy is applied to the crystallization of TTR complexes with curcumin and 16a-bromo-estradiol. Here we report the crystal structures with these compounds and with the ferulic acid that results from curcumin degradation

Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions

Synthesis and cycloxygenase inhibitory properties of new naphthalene-methylsulfonamido, naphthalene-methylsulfonyl and tetrahydronaphthalen-methylsulfonamido compounds

We synthesized a series of new naphthalene derivatives: naproxen- and 6-methoxy naphthalene acetic acid-like 1–5. In these compounds the carboxylic function, typical of the classical NSAIDs, was replaced by a methylsulfonamido (1, 2 and 6a–c) or methylsulfonyl (3–5) group present in some selective COX-2 inhibitors. We also synthesized compounds 7 and 8 in which the naphthalene portion was substituted by tetrahydronaphthalene ring. Some of the new compounds were assayed for their enzymatic inhibitory activity towards cycloxygenase enzymes. Compounds 4 and 6b, at a concentration of 10 µM exhibit percentage inhibition values of 65%, 50% and 29%, 87% towards COX-2 and COX-1, respectively. The substitution of carboxylic group with a mehylsulfonamido or a methylsulfonyl groups does not allow to direct the selectivity versus to cycloxygenase enzymes

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

FedDrive: Generalizing Federated Learning to Semantic Segmentation in Autonomous Driving

Semantic Segmentation is essential to make self-driving vehicles autonomous, enabling them to understand their surroundings by assigning individual pixels to known categories. However, it operates on sensible data collected from the users' cars; thus, protecting the clients' privacy becomes a primary concern. For similar reasons, Federated Learning has been recently introduced as a new machine learning paradigm aiming to learn a global model while preserving privacy and leveraging data on millions of remote devices. Despite several efforts on this topic, no work has explicitly addressed the challenges of federated learning in semantic segmentation for driving so far. To fill this gap, we propose FedDrive, a new benchmark consisting of three settings and two datasets, incorporating the real-world challenges of statistical heterogeneity and domain generalization. We benchmark state-of-the-art algorithms from the federated learning literature through an in-depth analysis, combining them with style transfer methods to improve their generalization ability. We demonstrate that correctly handling normalization statistics is crucial to deal with the aforementioned challenges. Furthermore, style transfer improves performance when dealing with significant appearance shifts. Official website: https://feddrive.github.io

N-(aroyl)-N-(arylmethyloxy)-α-alanines: selective inhibitors of aldose reductase

Aldose reductase (ALR2), a NADPH-dependent reductase, is the first and rate-limiting enzyme of the polyol pathway of glucose metabolism and is implicated in the pathogenesis of secondary diabetic complications. In the last decades, this enzyme has been targeted for inhibition but despite the numerous efforts made to identify potent and safe ALR2 inhibitors, many clinical candidates have been a failure. For this reason the research of new ALR2 inhibitors highly effective, selective and with suitable pharmacokinetic properties is still of great interest. In this paper some new N-(aroyl)-N-(arylmethyloxy)alanines have been synthesized and tested for their ability to inhibit ALR2. Some of the synthesized compounds exhibit IC50 in the low micromolar range and all have proved to be highly selective towards ALR2. The N-(aroyl)-N-(arylmethyloxy)-α-alanines are a promising starting point for the development of new ALR2 selective drugs with the aim of delaying the onset of diabetic complications

Targeting Different Transthyretin Binding Sites with Unusual Natural Compounds

Misfolding and aggregation of the transthyretin (TTR) protein leads to certain forms of amyloidosis. Some nutraceuticals, such as flavonoids and natural polyphenols, have recently been investigated as modulators of the self-assembly process of TTR, but they generally suffer from limited bioavailability. To discover innovative and more bioavailable natural compounds able to inhibit TTR amyloid formation, a docking study was performed using the crystallographic structure of TTR. This computational strategy was projected as an adhoc inspection of the possible relationship between binding site location and modulation of the assembly process; interactions with the as-yet-unexplored epigallocatechin gallate (EGCG) sites and with the thyroxine (T4) pocket were simultaneously analyzed. All the compounds studied seem to prefer the traditional T4 binding site, but some interesting results emerged from the screening of an in-house database, used for validating the computational protocol, and of the Herbal Ingredients Targets (HIT) catalogue available on the ZINC database

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

Intermittent versus continuous energy restriction on weight loss and cardiometabolic outcomes: A systematic review and meta-analysis of randomized controlled trials

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