Giulio Vistoli

VEGA – An open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming

In this paper we present the expandability and flexibility features of the VEGA program (downloadable free of charge at http://www.ddl.unimi.it), for the development of custom applications, using it as a multipurpose graphical environment. VEGA can be customized using both plug-in architecture and script programming. The first is useful to add new features and functions, using homemade routines, written with the VEGA Plug-in Development Kit (SDK). With the second approach it is possible to design scripts in VEGA, using the REBOL language, in order to (1) add new functions or customize existing ones; (2) automate common procedures; and (3) allow network communications, by creating a bridge between VEGA and other applications (or other PCs) through the TCP/IP protocol.

VEGA: a versatile program to convert, handle and visualize molecular structure on Windows-based PCs.

We here propose the program VEGA, that was developed to create a bridge between the most popular molecular software packages. In this tool some features are implemented some features to analyze, display and manage the three dimensional (3D) structure of the molecules. The most important features are (1) file format conversion (with assignment of the atom types and atomic charges), (2) surface calculation and (3) trajectory analysis. The executable and the source code can be free downloaded from [URL: see text].

Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation

Abstract Advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) have a pathogenetic role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis, and neurological disorders. AGEs and ALEs represent a quite complex class of compounds that are formed by different mechanisms, by heterogeneous precursors and that can be formed either exogenously or endogenously. There is a wide interest in AGEs and ALEs involving different aspects of research which are essentially focused on set-up and application of analytical strategies (1) to identify, characterize, and quantify AGEs and ALEs in different pathophysiological conditions; (2) to elucidate the molecular basis of their biological effects; and (3) to discover compounds able to inhibit AGEs/ALEs damaging effects not only as biological tools aimed at validating AGEs/ALEs as drug target, but also as promising drugs. All the above-mentioned research stages require a clear picture of the chemical formation of AGEs/ALEs but this is not simple, due to the complex and heterogeneous pathways, involving different precursors and mechanisms. In view of this intricate scenario, the aim of the present review is to group the main AGEs and ALEs and to describe, for each of them, the precursors and mechanisms of formation.

The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats

The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (β‐alanyl‐L‐histidine, L‐CAR) and of its enantiomer (β‐alanyl‐D‐histidine, D‐CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non‐diabetic, Zucker obese rat. The Zucker rats received a daily dose of L‐CAR or D‐CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly. At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L‐ and D‐CAR greatly reduced obese‐related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue. Because the protective effect elicited by L‐ and D‐CAR was almost superimposable, we conclude that the pharmacological action of L‐CAR is not due to a pro‐histaminic effect (D‐CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.

Reactions and enzymes in the metabolism of drugs and other xenobiotics

In this article, we offer an overview of the compared quantitative importance of biotransformation reactions in the metabolism of drugs and other xenobiotics, based on a meta-analysis of current research interests. Also, we assess the relative significance the enzyme (super)families or categories catalysing these reactions. We put the facts unveiled by the analysis into a drug discovery context and draw some implications. The results confirm the primary role of cytochrome P450-catalysed oxidations and UDP-glucuronosyl-catalysed glucuronidations, but they also document the marked significance of several other reactions. Thus, there is a need for several drug discovery scientists to better grasp the variety of drug metabolism reactions and enzymes and their consequences.

Albumin is the main nucleophilic target of human plasma : a protective role against pro-atherogenic electrophilic reactive carbonyl species?

The aim of this work was to study the metabolic fate of 4-hydroxy- trans-2-nonenal (HNE) in human plasma, which represents the main vascular site of reactive carbonyl species (RCS) formation and where the main pro-atherogenic target proteins are formed. When HNE was spiked in human plasma, it rapidly disappeared (within 40 s) and no phase I metabolites were detected, suggesting that the main fate of HNE is due to an adduction mechanism. HNE consumption was then monitored in two plasma fractions: low molecular weight plasma protein fractions (<10 kDa; LMWF) and high molecular weight plasma protein fractions (>10 kDa; HMWF). HNE was almost stable in LMWF, while in HMWF it was consumed by almost 70% within 5 min. Proteomics identified albumin (HSA) as the main protein target, as further confirmed by a significantly reduced HNE quenching of dealbuminated plasma. LC-ESI-MS/MS analysis identified Cys34 and Lys199 as the most reactive adduction sites of HSA, through the formation of a Michael and Schiff base adducts, respectively. The rate constant of HNE trapping by albumin was 50.61 +/- 1.89 M (-1) s (-1) and that of Cys34 (29.37 M (-1) s (-1)) was 1 order of magnitude higher with respect to that of GSH (3.81 +/- 0.17 M (-1) s (-1)), as explained by molecular modeling studies. In conclusion, we suggest that albumin, through nucleophilic residues, and in particular Cys34, can act as an endogenous detoxifying agent of circulating RCS.

Molecular strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced lipoxidation end products

Abstract The advanced glycoxidation end products (AGEs) and lipoxidation end products (ALEs) contribute to the development of diabetic complications and of other pathologies. The review discusses the possibilities of counteracting the formation and stimulating the degradation of these species by pharmaceuticals and natural compounds. The review discusses inhibitors of ALE and AGE formation, cross-link breakers, ALE/AGE elimination by enzymes and proteolytic systems, receptors for advanced glycation end products (RAGEs) and blockade of the ligand–RAGE axis.

Protein carbonylation : 2,4-dinitrophenylhydrazine reacts with both aldehydes/ketones and sulfenic acids

Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. beta-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.

Protein haptenation by amoxicillin: High resolution mass spectrometry analysis and identification of target proteins in serum

Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

Delta12-prostaglandin J2 as a product and ligand of human serum albumin: formation of an unusual covalent adduct at His146.

Human serum albumin (HSA), the most abundant protein in plasma, has a very unique function, catalyzing the conversion of prostaglandin J(2) (PGJ(2)), a dehydration product of PGD(2), to yield Delta(12)-PGJ(2). These PGD(2) metabolites are actively transported into cells and accumulated in the nuclei, where they act as potent inducers of cell growth inhibition and cell differentiation, and exhibit their own unique spectrum of biological effects. The facts that (i) arachidonic acid metabolites bind to human serum albumin (HSA) and the metabolism of these molecules is altered as a result of binding, (ii) HSA catalyzes the transformation of PGJ(2) into Delta(12)-PGJ(2), and (iii) Delta(12)-PGJ(2) is stable in serum suggest that HSA may bind and stabilize Delta(12)-PGJ(2) in a specific manner. A molecular interaction analysis using surface plasmon resonance (Biacore) indeed suggested the presence of a specific Delta(12)-PGJ(2)-binding site in HSA. To investigate the molecular details of the binding of this PGD(2) metabolite to albumin, we analyzed the cocrystal structure of the HSA-Delta(12)-PGJ(2)-myristate complex by X-ray crystallography and found that two Delta(12)-PGJ(2) molecules bind to a primary site in subdomain IB of the protein. The electron density results suggested that one of the two Delta(12)-PGJ(2) molecules that specifically bind to the site covalently interacted with a histidine residue (His146). Using nano-LC-MS/MS analysis of the HSA-Delta(12)-PGJ(2) complex, the formation of an unusual Delta(12)-PGJ(2)-histidine adduct at His146 was confirmed. Thus, our crystallographic and mass spectrometric analyses of the HSA-Delta(12)-PGJ(2) complex provided intriguing new insights into the molecular details of how this electrophilic ligand interacts with its primary producer and transporter.