Monounsaturated fatty acid ether oligomers formed during heating of virgin olive oil show agglutination activity against human red blood cells.

The present work focuses on the characterization of molecules formed when virgin olive oil is heated at 130 °C for 24 h open in air, which are found to be strong agglutinins. The hemagglutinating activity of the newly formed molecule isolated from the heated virgin olive oil sample was estimated against human red blood cells (RBCs). Dimers and polymers (high molecular weight molecules) were identified through thin layer chromatography (TLC) of the oil mixture. (1)H and (13)C nuclear magnetic resonance (NMR) and gas chromatography-mass spectroscopy (GC-MS) were the methods used for structural characterization. Among others, oligomerization of at least two monounsaturated fatty acids (FA) by an ether linkage between the hydrocarbon chains is involved. Light microscopy was used to characterize and visualize the agglutination process. Agglutination without fusion or lysis was observed. It was concluded that the heating of virgin olive oil open in air, among other effects, produces oligomerization as well as polymerization of unsaturated FA, possibly of monohydroxy, monounsaturated FA that is associated with strong hemagglutinating activity against human RBCs. The nutritional value and the effects on human health of such oligomers are not discussed in the literature and remain to be investigated.

Inhibition of secreted phospholipases A2 by 2-oxoamides based on α-amino acids: Synthesis, in vitro evaluation and molecular docking calculations.

Group IIA secreted phospholipase A2 (GIIA sPLA2) is a member of the mammalian sPLA2 enzyme family and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based on α-amino acids and the in vitro evaluation against three secreted sPLA2s (GIIA, GV and GX) are described. The long chain 2-oxoamide GK126 based on the amino acid (S)-leucine displayed inhibition of human and mouse GIIA sPLA2s (IC50 300nM and 180nM, respectively). It also inhibited human GV sPLA2 with similar potency, while it did not inhibit human GX sPLA2. The elucidation of the stereoelectronic characteristics that affect the in vitro activity of these compounds was achieved by using a combination of simulated annealing to sample low-energy conformations before the docking procedure, and molecular docking calculations.

Molecular docking and 3D-QSAR CoMFA studies on indole inhibitors of GIIA secreted phospholipase A(2).

Automated docking allowing a "protein-based" alignment was performed on a set of indole inhibitors of the GIIA secreted phospholipase A(2) (GIIA sPLA(2)). A correlation between the binding scores and the experimental inhibitory activity was observed (r(2) = 0.666, N = 34). All the indole inhibitors were docked in the active site of the GIIA sPLA(2) enzyme, and the best score docking pose of each inhibitor was used for the "protein-based" alignment of the compounds. A three-dimensional quantitative structure-activity relationship (3D-QSAR) model was then established using the comparative molecular field analysis (CoMFA) method. The set of 34 indole inhibitors was divided into two subsets: the training set, composed of 26 compounds, and the test set, consisting of eight compounds. The robustness and the predictive ability of the generated CoMFA model were examined by using the test set. A good correlation (r(2) = 0.997) between predicted and experimental inhibitory activity data allows the validation of the CoMFA model. Finally, the generated CoMFA model was used for the design and evaluation of new compounds. The new designed compounds exert improved predicted inhibitory activity and may be a target for the synthesis of new GIIA sPLA(2) indole inhibitors.

Hypertension study in anaesthetized rabbits: protocol proposal for AT1 antagonists screening.

INTRODUCTION: The aim of this study was to establish an optimized fast and safe protocol for the pharmacological screening of AT(1) antagonists. MATERIALS AND METHODS: The pharmaceutical prototype AT(1) antagonist losartan, its active metabolite EXP3174 and the synthetic compound MMK1 were analysed in order to validate the protocol. Ang II was continuously infused while the animals received the drugs in two procedures. RESULTS: In the post-treatment procedure drugs were administered either in a single bolus dose or in a sequential manner. When losartan was administered in a single bolus dose, efficacy was evident until the 7th min (p=0.012) whilst EXP3174 infusion extended the efficiency up to the end of the study (p=0.006). In addition, the sequential injections of losartan prolonged the inhibitory time interval until the end of the study (p=0.045). In the pre-treatment procedure, results suggested a dose-dependent inhibitory effect for both antagonists. The pressor response to Ang II was unchanged after MMK1 administration either in the post- or in the pre-treatment mode. CONCLUSIONS: The proposed protocol appears to be safe, simple and fast for the pharmacological screening of AT(1) antagonists and enables the evaluation of new antagonists using lower doses than any other reported in the literature.

Design of new secreted phospholipase A2 inhibitors based on docking calculations by modifying the pharmacophore segments of the FPL67047XX inhibitor.

Docking calculations that allow the estimation of the binding energy of small ligands in the GIIA sPLA(2) active site were used in a structure-based design protocol. Four GIIA sPLA(2) inhibitors co-crystallised with the enzyme, were used for examining the enzyme active site and for testing the FlexX in SYBYL 6.8 molecular docking program to reproduce the crystallographic experimental data. The FPL67047XX inhibitor was chosen as a prototype structure for applying free energy perturbation (FEP) studies. Structural modifications of the initial structure of the FPL67047XX inhibitor (IC(50) 0.013 microM) were performed in an effort to optimise the interactions in the GIIA sPLA(2) active site. The structural modifications were based on: (1) the exploration of absolute configuration (i.e. comparison of the binding score of (R)- and (S)-enantiomers); (2) bioisosterism (i.e. replacement of the carboxylate group with the bioisosteric sulphonate and phosphonate groups); (3) insertion of substituents that fit better in the active site. The generated new structures exhibited higher binding energy. Such structures may spark off the interest of medicinal chemists for synthesizing potentially more active GIIA sPLA(2) inhibitors.

A combined NMR and molecular dynamics simulation study to determine the conformational properties of agonists and antagonists against experimental autoimmune encephalomyelitis.

Myelin basic protein (MBP) is one of the best characterized autoantigens causing multiple sclerosis (MS), via a procedure that involves a stable formation of the trimolecular complex of a T-cell Receptor (TCR), an MBP epitope, and the receptor HLA-DR2b. Experimental autoimmune encephalomyelitis (EAE) is considered as an instructive model for MS in humans, and plenty of X-ray data is available for a number of EAE inducing peptide-receptor complexes. To date, though, there are no data available for complexes involving peptides reversing EAE, namely antagonists. Conformational properties of the EAE inducing epitope MBP(87-99) were analyzed in DMSO using the NOE connectivities and vicinal H(N)-H(alpha) coupling constants, and compared with the antagonist altered peptide ligands. A robust method, which is based on a combination of molecular dynamics and energy minimization, is proposed for identifying the putative bioactive conformations. Generated conformations are compared with the known X-ray structure of MBP(83-96) (human sequence numbering) in the HLA-DR2b complex. The structural motif for the agonist-antagonist activity is discussed.

The use of differential scanning calorimetry to study drug-membrane interactions.

Differential-scanning calorimetry is a thermodynamic technique widely used for studying drug-membrane interactions. This chapter provides practical examples on this topic, highlighting the caution to be taken in analyzing thermal data as well as scientific information that can be derived by the proper use of the technique. An example is given using model bilayers containing high concentration of the anesthetic steroid alphaxalone. It is shown that the breadth of the phase transitions and the maximum of the phase-transition temperature of the bilayer depend on the equilibration conditions before acquiring the thermal scan. In addition, the quality of the thermo-gram depends on its perturbation and incorporation effects; for dissecting these effects, a complementary technique such as solid-state nuclear magnetic resonance spectroscopy is necessary. Differential-scanning calorimetry is a useful technique to study the interdigitation effect of a drug by monitoring DeltaH changes. Cholesterol, a main constituent of membrane bilayers, appears to disrupt the interdigitating effect. In general, the thermal effects of the drug incorporated into a membrane bilayer depends on the drug stereoelectronic properties.

Molecular dynamics at the receptor level of immunodominant myelin basic protein epitope 87-99 implicated in multiple sclerosis and its antagonists altered peptide ligands: triggering of immune response.

This work reports molecular dynamics studies at the receptor level of the immunodominant myelin basic protein (MBP) epitope 87-99 implicated in multiple sclerosis, and its antagonists altered peptide ligands (APLs), namely [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99. The interaction of each peptide ligand with the receptor human leukocyte antigen HLA-DR2b was studied, starting from X-ray structure with pdb code: 1ymm. This is the first such study of APL-HLA-DR2b complexes, and hence the first attempt to gain a better understanding of the molecular recognition mechanisms that underlie TCR antagonism by these APLs. The amino acids His88 and Phe89 serve as T-cell receptor (TCR) anchors in the formation of the trimolecular complex TCR-peptide-HLA-DR2b, where the TCR binds in a diagonal, off-centered mode to the peptide-HLA complex. The present findings indicate that these two amino acids have a different orientation in the APLs [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99: His88 and Phe89 remain buried in HLA grooves and are not available for interaction with the TCR. We propose that this different topology could provide a possible mechanism of action for TCR antagonism.

Comparison of proposed putative active conformations of myelin basic protein epitope 87-99 linear altered peptide ligands by spectroscopic and modelling studies: the role of positions 91 and 96 in T-cell receptor activation.

This work proposes a structural motif for the inhibition of experimental autoimmune encephalomyelitis (EAE) by the linear altered peptide ligands (APLs) [Ala91,96] MBP87-99 and [Arg91,Ala96] MBP87-99 of myelin basic protein. Molecular dynamics was applied to reveal distinct populations of EAE antagonist [Ala91,96] MBP87-99 in solution, in agreement with NOE data. The combination of the theoretical and experimental results led to the identification of a putative active conformation. This approach is of value as no crystallographic data is available for the APL-receptor complex. TCR contact residue Phe89 has an altered topology in the putative bioactive conformations of both APLs with respect to the native peptide, as found via crystallography; it is no longer prominent and solvent exposed. It is proposed that the antagonistic activity of the APLs is due to their binding to MHC, preventing the binding of self-myelin epitopes, with the absence of an immunologic response as the loss of some interactions with the TCR hinders activation of T-cells.

Authenticity of the traditional cypriot spirit "zivania" on the basis of 1h NMR spectroscopy diagnostic parameters and statistical analysis.

A previous publication (Kokkinofta et al. J. Agric. Food Chem. 2003, 51, 6233-6239) discussed the use of inductively coupled plasma spectroscopy to differentiate between the traditional Cypriot alcoholic beverage zivania and other spirits similar in alcoholic content collected from different countries. In the present paper (1)H NMR spectroscopy is applied to confirm the previous conclusions and to obtain additional physical-chemical characteristics that may be used to differentiate zivania from other similar beverages. NMR spectroscopy gave a satisfactory degree of prediction and classification between zivanias and other distillings. The validity of quantification of the method was tested using comparative GC data. It appears that chemical analysis can be very helpful for identifying the unique geological and climatic conditions existing in the island of Cyprus that lead to an authentic product.