Anna Ramunno

Interaction between Alzheimer's Aβ(25–35) peptide and phospholipid bilayers: The role of cholesterol

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimers disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).

Progresses in the pursuit of aldose reductase inhibitors: The structure-based lead optimization step

Aldose reductase (ALR2) is a crucial enzyme in the development of the major complications of diabetes mellitus. Very recently it has been demonstrated that the ARL2 inhibitor, fidarestat, significantly prevents inflammatory signals (TNF-α, LPS) that cause cancer (colon, breast, prostate and lung), metastasis, asthma, and other inflammatory diseases. Currently, fidarestat is in phase III clinical trial for diabetic neuropathy and was found to be safe. Thus the finding of novel, potent ARL2 inhibitors is today more than in the past in great demand as they can pave the way for a novel therapeutic approach for a number of diseases besides the diabetes. Herein, starting from the virtual screening-derived ALR2 inhibitor S12728 (1), a rational receptor-based lead optimization has been undertaken. The design and synthetic efforts here reported led to the discovery of several new compounds endowed with low micromolar/submicromolar activities.

Interaction of a beta-sheet breaker peptide with lipid membranes.

Aggregation of β‐amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimers disease. An attractive therapeutic strategy for Alzheimers disease is the inhibition of the soluble β‐amyloid aggregation using synthetic β‐sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β‐sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β‐sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β‐sheet breaker peptide acetyl‐LPFFD‐amide, iAβ5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C‐ and at the N‐terminus or phospholipids spin‐labeled in different positions of the acyl chain. Our results show that iAβ5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright

Specific Targeting of Highly Conserved Residues in the HIV-1 Reverse Transcriptase Primer Grip Region. 2. Stereoselective Interaction to Overcome the Effects of Drug Resistant Mutations

Starting from the prototypic compound 4, we describe new, potent, and broad-spectrum pyrrolobenzo(pyrido)oxazepinones antivirals. A biochemical and enzymological investigation was performed for defining their mechanism of inhibition at either recombinant HIV-1 RT wild type and non-nucleoside reverse transcriptase inhibitors (NNRTIs)-resistant mutants. For the novel compounds (S)-(+)-5 and (S)-(-)-7, a clear-cut stereoselective mechanism of enzyme inhibition was found. Molecular modeling studies were performed for revealing the underpinnings of this behavior.

Novel sterically hindered cannabinoid CB1 receptor ligands

In the present study, 11 novel N-(3,3-diphenyl)propyl-2,2-diphenylacetamide derivatives (4a-d and 9a-g) and six triphenylacetamides (10a-c and 11a-c) were synthesized and tested as ligands of cannabinoid CB(1) and CB(2) receptors. All compounds exhibited affinity for CB(1) and CB(2) receptors. Four compounds (4b, 9a, 9b, and 11a) showed selectivity for CB(1) versus CB(2) receptors, although only the N-(3,3-diphenyl)propyl-2,2-diphenylacetamide (4b) can be considered a potent CB(1) ligand (K(i)=58 nM). It was 140-fold selective over CB(2) receptors (K(i)=7800 nM) and behaved as an inverse agonist by stimulating forskolin-induced cAMP formation in mouse N18TG2 neuroblastoma cells. This compound is the first of a novel class of tetraphenyl CB(1) ligands that, in view of its easy synthesis and high affinity for CB(1) receptors and despite its sterical hindrance, will be useful for the design of new blockers of this therapeutically exploitable receptor type.

Antioxidant Activity of Diphenylpropionamide Derivatives: Synthesis, Biological Evaluation and Computational Analysis

We report the synthesis, antioxidant and antiproliferative activity and a QSAR analysis of synthetic diphenylpropionamide derivatives. Synthesis of these compounds was achieved by direct condensation of 2,2- and 3,3-diphenylpropionic acid and appropriate amines using 1-propylphoshonic acid cyclic anhydride (PPAA) as catalyst. Compound structures were elucidated by NMR analysis and their melting points were measured. The in vitro antioxidant activity of these compounds was tested by evaluating the amount of scavenged ABTS radical and estimating ROS and NO production in LPS stimulated J774.A1 macrophages. All compounds were tested for their effect on viability of cells and results demonstrated that they are not toxic towards the cell lines used. The cytotoxic activity of all compounds was evaluated by a Brine Shrimp Test.

Anti-Inflammatory, Antioxidant and Crystallographic Studies of N-Palmitoyl-ethanol Amine (PEA) Derivatives

N-Palmitoyl-ethanolamine (PEA) is an anti-inflammatory component of egg yolk that is usually employed for the prevention of respiratory apparatus virus infection and then frequently used for its efficient anti-inflammatory and analgesic effects in experimental models of visceral, neuropathic, and inflammatory diseases. Nevertheless, data of its use in animal or human therapy are still scarce and further studies are needed. Herein, we report the biological evaluation of a small library of N-palmitoyl-ethanolamine analogues or derivatives, characterized by a protected acid function (either as palmitoyl amides or hexadecyl esters), useful to decrease their hydrolysis rate in vitro and prolong their biological activity. Two of these compounds—namely phenyl-carbamic acid hexadecyl ester (4) and 2-methyl-pentadecanoic acid (4-nitro-phenyl)-amide (5)—have shown good anti-inflammatory and antioxidant properties, without affecting the viability of J774A.1 macrophages. Finally, crystals suitable for X-ray analysis of compound 4 have been obtained, and its solved crystal structure is here reported. Our outcomes may be helpful for a rational drug design based on new PEA analogues/derivatives with improved biological properties.

Activation of the Wnt pathway by small peptides: rational design, synthesis and biological evaluation

A computational analysis of the X‐ray structure of the low‐density lipoprotein receptor‐related protein 6 (LRP6) with the Dickkopf‐1 (DKK1) C‐terminal fragment has allowed us to rationally design a small set of decapeptides. These compounds behave as agonists of the canonical Wnt pathway in the micromolar range when tested on a dual luciferase Wnt functional assay in glioblastoma cells. Two of the oligopeptides showed a lack of cytotoxicity in human primary osteoblasts isolated from sponge bone tissue (femoral heads or knees of elderly patients). According to the mechanism of action, the studies revealed a dose‐ and time‐dependent increase in the viability of human osteoblasts. These results may indicate a potential therapeutic application of this class of compounds in the treatment of bone diseases related to aging, such as osteoporosis.