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

Sphingosine 1-phosphate receptor 1 is required for MMP-2 function in bone marrow mesenchymal stromal cells: implications for cytoskeleton assembly and proliferation

Bone marrow-derived mesenchymal stromal cell- (BM-MSC-) based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP-) 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P) and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers) and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia), S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs' ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair

The Impairment of CD8 Responses Limits the Selection of Escape Mutations in Acute Hepatitis C Virus Infection

Abstract Evasion from protective CD8 responses by mutations within immunodominant epitopes represents a potential strategy of HCV persistence. To investigate the pathogenetic relevance of this mechanism, a careful search for immunodominant CD8 epitopes was conducted in six patients with chronic evolution of HCV infection by analyzing their global CD8 response with a panel of overlapping synthetic peptides covering the overall HCV sequence and by studying the CD8 frequency by tetramer staining. Immunodominant responses were followed longitudinally from the time of acute onset in relation to the evolution of the epitopic sequences. Although intensity of CD8 responses and frequency of HCV-specific CD8 cells declined over time in all patients, mutations emerged in only three of the six acute patients studied. Variant sequences were less efficiently recognized by CD8 cells than parental epitopes and were poorly efficient in inducing a CD8 response in vitro. CD8 epitopes undergoing mutations were targeted by high avidity CD8 cells more efficient in effector function. Our data support the view that immunodominant CD8 responses are affected by inhibitory mechanisms operating early after infection and that the emergence of escape mutations represents an additional mechanism of virus evasion from those CD8 responses that are functionally preserved

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

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

Copper mediated amyloid-β binding to Transthyretin

Transthyretin (TTR), a homotetrameric protein that transports thyroxine and retinol both in plasma and in cerebrospinal (CSF) fluid provides a natural protective response against Alzheimer’s disease (AD), modulates amyloid-β (Aβ) deposition by direct interaction and co-localizes with Aβ in plaques. TTR levels are lower in the CSF of AD patients. Zn2+, Mn2+and Fe2+transform TTR into a protease able to cleave Aβ. To explain these activities, monomer dissociation or conformational changes have been suggested. Here, we report that when TTR crystals are exposed to copper or iron salts, the tetramer undergoes a significant conformational change that alters the dimer-dimer interface and rearranges residues implicated in TTR’s ability to neutralize Aβ. We also describe the conformational changes in TTR upon the binding of the various metal ions. Furthermore, using bio-layer interferometry (BLI) with immobilized Aβ(1–28), we observe the binding of TTR only in the presence of copper. Such Cu2+-dependent binding suggests a recognition mechanism whereby Cu2+modulates both the TTR conformation, induces a complementary Aβ structure and may participate in the interaction. Cu2+-soaked TTR crystals show a conformation different from that induced by Fe2+, and intriguingly, TTR crystals grown in presence of Aβ(1–28) show different positions for the copper sites from those grown its absence

Mesenchymal stromal cells exert a stimulatory effect on skeletal myoblast proliferation through the release of Sphingosine 1-phosphate (S1P)

Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration [1]. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses [2], is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by MSCconditioned medium, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration