Bartolomé Vilanova

The pyridoxamine action on Amadori compounds: A reexamination of its scavenging capacity and chelating effect.

Amadori compounds act as precursors in the formation of advanced glycation end products (AGEs) by non-enzymatic protein glycation, which are involved in ensuing protein damage. Pyridoxamine is a potent drug against protein glycation, and can act on several pathways in the glycation process. Nevertheless, the pyridoxamine inhibition action on Amadori compounds oxidation is still unclear. In this work, we have studied the Schiff base formation between pyridoxamine and various Amadori models at pH 7.4 at 37 degrees C in the presence of NaCNBH(3). We detected an adduct formation, which suggests that pyridoxamine reacts with the carbonyl group in Amadori compounds. The significance of this mechanism is tested by comparison of the obtained kinetics rate constants with that obtained for 4-(aminomethyl)-pyridine, a structural analogue of pyridoxamine without post-Amadori action. We also study the chelating effect of pyridoxamine on metal ions. We have determined the complexation equilibrium constants between pyridoxamine, N-(1-deoxy-d-fructos-1-yl)-l-tryptophan, aminoguanidine, and ascorbic acid in the presence of Zn(2+). The results show that the strong stability of pyridoxamine complexes is the key in its post-Amadori inhibition action. On the other hand results explain the lack of inhibition of aminoguanidine (a glycation inhibitor) in the post-Amadori reactions.

Mechanistic insights in glycation-induced protein aggregation.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

A comparative study of the chemical reactivity of pyridoxamine, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds.

Pyridoxamine (PM) has long been known to inhibit protein glycation via various mechanisms of action. One such mechanism involves the scavenging of carbonyl compounds with glycating ability. Despite the abundant literature on this topic, few quantitative kinetic studies on the processes involved have been reported. In this work, we conducted a comparative kinetic study under physiological pH and temperature conditions of the reactions of PM, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds (viz. aldehydes, α-oxoaldehydes and ketones). The microscopic formation rate constants for Schiff bases of PM and various carbonyl compounds, k1, are of the same order of magnitude as those for the Schiff bases of Ac-Phe-Lys. However, because PM exhibits a higher proportion of reactive form at physiological pH, its observed second-order rate constant is ca. five times greater than that for Ac-Phe-Lys. That could explain PM ability to compete with amino residues in protein glycation. On the other hand, the observed formation rate constant for thiohemiacetals is four orders of magnitude greater than the formation constants for the Schiff bases of PM, which excludes PM as a competitive inhibitor of Cys residues in protein glycation.

Unexpected isomeric equilibrium in pyridoxamine Schiff bases

Pyridoxamine is a vitamin B(6) derivative involved in biological reactions such as transamination, and can also act as inhibitor in protein glycation. In both cases, it has been reported that Schiff base formation between pyridoxamine and carbonyl compounds is the main step. Nevertheless, few studies on the Schiff base formation have been reported to date. In this work, we conduct a comparative study of the reaction of pyridoxamine and 4-picolylamin (a pyridoxamine analog) with various carbonyl compounds including propanal, formaldehyde and pyruvic acid. Based on the results, 4-picolylamin forms a Schiff base as end-product of its reactions with propanal and pyruvic acid, but a carbinolamine with formaldehyde. On the other hand, pyridoxamine forms a Schiff base with the three reagents, but the end-product is in equilibrium with its hemiaminal form, which results from the attack of the phenolate ion of the pyridine ring on the imine carbon. This isomeric equilibrium should be considered in studying reactions involving amine derivatives of vitamin B(6).

Kinetic Study of the Reaction of Glycolaldehyde with Two Glycation Target Models

We have studied the reactivity of glycolaldehyde (GLA) with N‐acetyl‐Cys and N‐acetyl‐Phe‐Lys at physiological conditions of pH and temperature. The reaction between the N‐Ac‐Phe‐Lys and GLA was studied in the presence of NaCNBH3 and then by using high‐performance liquid chromatography (HPLC)‐UV/Vis. The reaction between N‐Ac‐Cys and GLA was followed by stopped‐flow spectroscopy with UV/Vis detection. Both the reduced Schiff base and thiohemiacetal were identified by 1H‐NMR and HPLC‐mass spectrometry detection. The kinetic rate constant for the thiohemiacetal formation is four orders of magnitude higher than that for the Schiff base formation. This result suggests that the thiol group represents the most important target in protein glycation.

Phenol Group in Pyridoxamine Acts as a Stabilizing Element for Its Carbinolamines and Schiff Bases

Pyridoxamine (PM), a natural derivative of vitamin B6, possesses a high biological and biomedical significance by virtue of its acting as enzyme cofactor in amino acid metabolism and as inhibitor in the nonenzymatic glycation of proteins. Both types of processes require the initial formation of a Schiff base. In this work, we used NMR spectroscopy to study the formation mechanism for a Schiff base between PM and formaldehyde (FA). This allowed the Schiff base and an intermediate carbinolamine (CA) to be detected. The Schiff base was found to be in isomeric equilibrium with a hemiaminal (HE) form. The formation equilibrium constants for the CA and HE over the pD range of 6.0–13.0 were determined and compared with those for the reaction between 4‐picolylamine (PAM) and formaldehyde (FA). The comparison revealed a strong influence of the phenol group on the equilibrium constant. Based on the results, the phenol group in PM is a key structural element towards stabilizing the resulting carbinolamine and Schiff base.

Alkaline hydrolysis of N-methylazetidin-2-one. Hydration effects

Abstract Semiempirical calculations (PM3) have been used to investigate the reaction mechanism (BAC2) of the alkaline hydrolysis of N-methylazetidin-2-one. This mechanism involves the nucleophilic attack of a hydroxyl ion on the carbonyl carbon to give a tetrahedral complex followed by cleavage of the CN bond and proton transfer to form the final product. The influence of the solvent in this process has been analyzed using the supermolecular approach with a water solvation sphere of 20 molecules around the solute. The results obtained have been compared with those based on a continuum treatment of the solvent with semiempirical and ab initio methodology. The potential barrier of 17.5 kcal mol−1 due to the attack of the nucleophile is very close to the experimental value (16.1 kcal mol−1) and the final product is about 52 and 27 kcal mol−1 more stable than the reactives and the tetrahedral intermediate, respectively.

Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

Protein aggregation with the concomitant formation of amyloid fibrils is related to several neurodegenerative diseases, but also to non-neuropathic amyloidogenic diseases and non-neurophatic systemic amyloidosis. Lysozyme is the protein involved in the latter, and it is widely used as a model system to study the mechanisms underlying fibril formation and its inhibition. Several phenolic compounds have been reported as inhibitors of fibril formation. However, the anti-aggregating capacity of other heteroaromatic compounds has not been studied in any depth. We have screened the capacity of eleven different hydroxypyridines to affect the acid-induced fibrillization of hen lysozyme. Although most of the tested hydroxypyridines alter the fibrillation kinetics of HEWL, only 3-hydroxy-2-methylpyridine, 3-hydroxy-6-methylpyridine and 3-hydroxy-2,6-dimethylpyridine completely abolish fibril formation. Different biophysical techniques and several theoretical approaches are combined to elucidate their mechanism of action. O-methylated 3-hydroxypyridines bind non-cooperatively to two distinct but amyloidogenic regions of monomeric lysozyme. This stabilises the protein structure, as evidenced by enhanced thermal stability, and results in the inhibition of the conformational transition that precedes fibril assembly. Our results point to o-methylated 3-hydroxypyridines as a promising molecular scaffold for the future development of novel fibrillization inhibitors.

Molecular modelling studies on Henry–Michaelis complexes of a class-C β-lactamase and β-lactam compounds

Abstract Molecular mechanics calculations based on the AMBER force field were used to construct molecular models for the Henry–Michaelis complexes of Enterobacter cloacae P99, a class C β-lactamase, with various β-lactam antibiotics. The results confirm the high significance of the carboxyl group borne by the β-lactam to the binding interaction with the enzymes active site. The differential conformation adopted by the carboxyl group in penicillins and cephalosporins is clearly reflected in the behaviour of the complexes formed. Thus, penicillins and cephalosporins form a strong hydrogen bond between one of the oxygen atoms in the carboxyl group and the hydroxyl group in the side chain of Thr-316 and Ser-318, respectively. This differential interaction of the carboxyl group in the two types of antibiotic also results in differential interactions of the oxygen atom in the β-lactam carbonyl with the Ser-64 and Ser-318 enzyme residues.

Ab initio study of the alkaline hydrolysis of a thio-β-lactam structure

Abstract The alkaline hydrolysis of a thio-β-lactam in the gas phase was examined in the light of RHF and DFT ab initio calculations. The solvent effect was considered via IPCM computations. The tetrahedral intermediate for the thio-β-lactam studied is unstable, so the compound evolves directly to the corresponding thio-azethidin-2-one open ring with cleavage of the C–S bond. The end-products obtained bear a carbamate group, which suggests that the thio-β-lactam might be an effective inhibitor for β-lactamases.