Luca Massaccesi

Insight into the binding interactions of CYP450 aromatase inhibitors with their target enzyme: A combined molecular docking and molecular dynamics study

AbstractCYP450 aromatase catalyzes the terminal and rate-determining step in estrogen synthesis, the aromatization of androgens, and its inhibition is an efficient approach to treating estrogen-dependent breast cancer. Insight into the molecular basis of the interaction at the catalytic site between CYP450 aromatase inhibitors and the enzyme itself is required in order to design new and more active compounds. Hence, a combined molecular docking–molecular dynamics study was carried out to obtain the structure of the lowest energy association complexes of aromatase with some third-generation aromatase inhibitors (AIs) and with other novel synthesized letrozole-derived compounds which showed high in vitro activity. The results obtained clearly demonstrate the role of the pharmacophore groups present in the azaheterocyclic inhibitors (NSAIs)—namely the triazolic ring and highly functionalized aromatic moieties carrying H-bond donor or acceptor groups. In particular, it was pointed out that all of them can contribute to inhibition activity by interacting with residues of the catalytic cleft, but the amino acids involved are different for each compound, even if they belong to the same class. Furthermore, the azaheterocyclic group strongly coordinates with the Fe(II) of heme cysteinate in the most active NSAI complexes, while it prefers to adopt another orientation in less active ones. Figure3D structure of the Michaelis complex for Androstenedione-Aromatase

Insights into the influence of 5-HT2c aminoacidic variants with the inhibitory action of serotonin inverse agonists and antagonists

AbstractSpecific modulation of serotonin 5-HT2C G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. The different efficacy of drugs targeting these receptors are due to the presence of genetic variants in population and this variability is still hard to predict. Therefore, in order to administer the more suitable drug, taking into account patient genotype, it is necessary to know the molecular effects of its gene nucleotide variations. In this work, starting from an accurate 3D model of 5-HT2C, we focus on the prediction of the possible effect of some single nucleotide polymorphisms (SNPs) producing amino acidic changes in proximity of the 5-HT2C ligand binding site. Particularly we chose a set of 5-HT2C inverse agonists and antagonists which have high inhibitory activity. After prediction of the structures of the receptor-ligand complexes using molecular docking tools, we performed full atom molecular dynamics simulations in explicit lipid bilayer monitoring the interactions between ligands and trans-membrane helices of the receptor, trying to infer relations with their biological activity. Serotonin, as the natural ligand was chosen as reference compound to advance a hypothesis able to explain the receptor inhibition mechanism. Indeed we observed a different behavior between the antagonists and inverse agonist with respect to serotonin or unbounded receptor, which could be responsible, even if not directly, of receptor’s inactivation. Furthermore, we analyzed five aminoacidic variants of 5HT2C receptor observing alterations in the interactions between ligands and receptor which give rise to changes of free energy values for every complex considered. FigureAntagonist and inverse agonist binding sites in 5-HT2C wild type and its SNP variants

In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats.

The present work was designed to study the mechanisms associated with Nurr1 modulation following early life permethrin (PERM) treatment during rats life span. Here we demonstrate that PERM exposure in rats, at a dose close to No Observed Adverse Effect Level (NOAEL) for 15days during neonatal brain development leads to its accumulation long after exposure. In striatum from adolescent rats we detected an increase in DNA methyltransferases (DNMTs) such as DNMT1, DNMT3a, Tyrosine hydroxylase, monomeric and aggregated α-synuclein protein levels. Adult rats showed enhanced DNMT3b and α-synuclein aggregation compared to the control group, while with aging a significant decrease in all biomarkers studied was observed. No changes in Nurr1 promoter methylation in adolescent, adult and old rats were found. In silico studies showed clear evidence of a strong binding interaction between PERM and its metabolite 3-phenoxybenzoic acid with the nuclear orphan receptor Nurr1. These findings suggest that an additional interference with the dopaminergic neuron pathway could occur in situ during PERM accumulation in brain. Therefore, Nurr1 modulation in early life PERM-treated rats, depends on age-related adaptive responses in animals.

Salts Influence Cathechins and Flavonoids Encapsulation in Liposomes: a Molecular Dynamics Investigation.

Cathechins and flavonoids are responsible of numerous health benefits. Two of the most representatives’ compounds for their antioxidant and therapeutic effects are Epigallocatechin 3‐Gallate (EGCG), from green tea extracts, and morelloflavone (MF), from Garcinia dulcis. Here we explore, by atomistic Molecular Dynamics simulations, how EGCG and MF interact with lipid bilayers and we show the salts’ influence on their encapsulation degree in neutral liposomes. As a result, we found out that EGCGs naturally bind to the hydrophilic regions of phospholipids, positioning themselves mostly at the interface between water and lipid phases. The presence of a salt clearly influences the EGCG molecules’ absorption and the total effect depends strongly on the salt nature and concentration. Beside, for MF, we observed a high stability of the intermolecular MFs aggregates in water that strongly penalizes the flavonoids interaction with the lipid polar heads. However, salts can influence MF′s liposomal penetration, even if they are not able to promote completely its absorption inside the bilayer. For both compounds, the increase of penetration is more marked in presence of magnesium chloride, whilst calcium chloride showed the opposite effect.

A possible S-glutathionylation of specific proteins by glyoxalase II: An in vitro and in silico study: Glo2 enzyme promotes S-glutathionylation

Glyoxalase II, the second of 2 enzymes in the glyoxalase system, is a hydroxyacylglutathione hydrolase that catalyses the hydrolysis of S‐d‐lactoylglutathione to form d‐lactic acid and glutathione, which is released from the active site. The tripeptide glutathione is the major sulfhydryl antioxidant and has been shown to control several functions, including S‐glutathionylation of proteins. S‐Glutathionylation is a way for the cells to store reduced glutathione during oxidative stress, or to protect protein thiol groups from irreversible oxidation, and few enzymes involved in protein S‐glutathionylation have been found to date. In this work, the enzyme glyoxalase II and its substrate S‐d‐lactoylglutathione were incubated with malate dehydrogenase or with actin, resulting in a glutathionylation reaction. Glyoxalase II was also submitted to docking studies. Computational data presented a high propensity of the enzyme to interact with malate dehydrogenase or actin through its catalytic site and further in silico investigation showed a high folding stability of glyoxalase II toward its own reaction product glutathione both protonated and unprotonated. This study suggests that glyoxalase II, through a specific interaction of its catalytic site with target proteins, could be able to perform a rapid and specific protein S‐glutathionylation using its natural substrate S‐d‐lactoylglutathione.

Liposomal Formulations for an Efficient Encapsulation of Epigallocatechin-3-Gallate: An In-Silico/Experimental Approach

As a part of research project aimed to optimize antioxidant delivery, here we studied the influence of both salts and lipid matrix composition on the interaction of epigallocatechin-3-gallate (EGCG) with bilayer leaflets. Thus, we combined in silico and experimental methods to study the ability of neutral and anionic vesicles to encapsulate EGCG in the presence of Ca2+ and Mg2+ divalent salts. Experimental and in silico results show a very high correlation, thus confirming the efficiency of the developed methodology. In particular, we found out that the presence of calcium ions hinders the insertion of EGCG in the liposome bilayer in both neutral and anionic systems. On the contrary, the presence of MgCl2 improves the insertion degree of EGCG molecules respect to the liposomes without divalent salts. The best and most efficient salt concentration is that corresponding to a 5:1 molar ratio between Mg2+ and EGCG, in both neutral and anionic vesicles. Concerning the lipid matrix composition, the anionic one results in better promotion of the catechin insertion within the bilayer since experimentally we achieved 100% EGCG encapsulation in the lipid carrier in the presence of a 5:1 molar ratio of magnesium. Thus, the combination of this anionic liposomal formulation with magnesium chloride, avoids time-consuming separation steps of unentrapped active principle and appears particularly suitable for EGCG delivery applications.

Recent Advances in Computational Simulations of Lipid Bilayer Based Molecular Systems

Abstract: Computer simulations in lipid bilayers research has become prominent for the last couple of decades. As computational resources became more available to the scientific community, simulations play an increasingly important role in understanding the processes that take place in and across cell membranes. The scientific interest is strictly related to the Biological importance of the Biomembranes, which act as barriers separating cell’s internal environment from the external one. Membranes are selectively permeable, and thus they actively participate in the movement control of compounds into and outside cells. These membranes have an heterogeneous complex composition and they include many different lipids together with proteins, steroids, carbohydrates and other membrane-associated molecules. Each of these compounds are involved in a great number of cellular processes and thus, membranes exist as dynamic structures. As a consequence, the understanding of biomembrane functioning requires the knowledge of chemical-physical behavior of lipid bilayers and it represents a great challenge in biophysical and medical sciences. In the last decades, molecular dynamics (MD) simulations have become one of the most useful tool in the in silico investigations of molecular structures; in fact, such computations provide structural dynamical information which is essential and hardly obtained by experimental methods; furthermore, it furnishes a system real-time imaging at atomistic-level resolution. In this chapter, we want to point out the recent advances in computer simulations in the field of lipid bilayers and proteins-lipid bilayers systems during the last few years, by covering several selected subjects such as state of art in ad hoc force fields’ development, Cholesterol induced effects on structure and properties of the bilayer, mixed composition lipid matrix, and biomolecular application of coarsegrained models.

Glyoxalase II promotes "in vitro" S-glutathionylation

S-glutathionylation involves the reversible formation of a mix disulphide-bridge between specific cysteine and a molecule of glutathione, the major non-protein antioxidant compound in the cell. Mechanisms of protein S-glutathionylation are far to be completely understood and several reactions can promote it, either spontaneously or catalyzed. For the first time Glo II enzyme was studied as a new potential candidate to promote S-glutathionylation. To demonstrate its active involvement in protein glutathionylation were used actin, malate dehydrogenase and GAPDH purified proteins, which are known to be glutathionylated, for in vitro experiments..This work shows active involvement of cytosolic Glo II for in vitro protein S-glutathionylation. To confirm the role of Glo II, preliminary protein-protein docking studies was performed between Glo II and human actin. The data showed a high propensity to aggregate with other proteins through its catalytic site Further, in silico investigation of Glo II stability and behavior, conducted through full atom molecular dynamics simulations, showed an high folding stability together with a great affinity towards its own reaction product glutathione both protonated (GSH) and unprotonated (GS(-)). These studies, revealed that GloII, using its natural substrate SLG, allow a rapid and specific protein-SSG formation, leading enzymatic regulation of S-glutathionylation in proteins of different origin and cellular compartmentalization.