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The impact of species-dependent differences between human and rat MAO B on inhibitor screening was evidenced for two classes of compounds, coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives. All examined compounds have shown a greater inhibitor potency toward human MAO B than toward rat MAO B. Moreover, no correlation was found between human and rat pIC(50) values. These divergences have important implications for the design and development of drugs involved in the MAO B metabolic pathway, suggesting that results obtained using rat enzyme cannot be extrapolated to human CNS, a priori. Indeed, the selection of a hit compound for lead generation could be different using human rather than rat enzyme. Moreover, the influence of substituents on the in vitro inhibition of human MAO B was markedly different between homogeneous series of coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives, suggesting different binding modes, a hypothesis clearly supported by molecular docking simulations of inhibitors into the active site of human MAO B.

Two new xanthone glycosides, corymbiferin 3-O-beta-D-glucopyranoside (1) and swertiabisxanthone-I 8'-O-beta- d-glucopyranoside (2), were isolated from Gentianella amarella ssp. acuta, along with eight known xanthones: triptexanthoside C, veratriloside, corymbiferin 1-O-glucoside, swertianolin, norswertianolin, swertiabisxanthone-I, bellidin, and bellidifolin, four of them identified for the first time in G. amarella ssp. acuta. The isolation was conducted mainly by centrifugal partition chromatography, and the structures of the isolated compounds were established on the basis of spectrometric data including 2D NMR and mass spectrometry. Xanthones were weakly active against acetylcholinesterase (AChE), except triptexanthoside C, which inhibited AChE with an IC(50) of 13.8 +/- 1.6 microM. Some compounds were active against monoamine oxidases (MAO): bellidin and bellidifolin showed interesting inhibitory activity of MAO A, while swertianolin, the 8-O-glucopyranoside form of bellidifolin, gave 93.6% inhibition of MAO B activity at 10(-5) M.

GOLD is a molecular docking software widely used in drug design. In the initial steps of docking, it creates a list of hydrophobic fitting points inside protein cavities that steer the positioning of ligand hydrophobic moieties. These points are generated based on the Lennard-Jones potential between a carbon probe and each atom of the residues delimitating the binding site. To thoroughly describe hydrophobic regions in protein pockets and properly guide ligand hydrophobic moieties toward favorable areas, an in-house tool, the MLP filter, was developed and herein applied. This strategy only retains GOLD hydrophobic fitting points that match the rigorous definition of hydrophobicity given by the molecular lipophilicity potential (MLP), a molecular interaction field that relies on an atomic fragmental system based on 1-octanol/water experimental partition coefficients (log P(oct)). MLP computations in the binding sites of crystallographic protein structures revealed that a significant number of points considered hydrophobic by GOLD were actually polar according to the MLP definition of hydrophobicity. To examine the impact of this new tool, ligand-protein complexes from the Astex Diverse Set and the PDB bind core database were redocked with and without the use of the MLP filter. Reliable docking results were obtained by using the MLP filter that increased the quality of docking in nonpolar cavities and outperformed the standard GOLD docking approach.

Aging related neurodegenerative disorders such as Parkinson disease (PD) and Alzheimer's disease (AD) are the result of multiple pathophysiological pathways that contribute to the neurodegenerative cascade. Hence, multifunctional drug candidates able to interact with several targets are of great interest for the treatment of such diseases. Therefore, an experimental and virtual screening pathway to generate multifunctional hits showing promise for the treatment of PD or AD was suggested. Moreover, suitable experimental and virtual screening methods to rapidly test pre-focused compound libraries were developed and validated. In particular, the screening was focused on potential inhibitors of acetylcholinesterase (AChE) and monoamine oxidase B (MAO B) using a combination of in vitro enzymatic tests, docking and scoring approaches and refined molecular modeling tools.

Haemolytic Uraemic Syndrome associated with Streptococcus pneumoniae infections (SP-HUS) is a clinically well-known entity that generally affects infants, and could have a worse prognosis than HUS associated to E. coli infections. It has been assumed that complement genetic variants associated with primary atypical HUS cases (aHUS) do not contribute to SP-HUS, which is solely attributed to the action of the pneumococcal neuraminidase on the host cellular surfaces. We previously identified complement pathogenic variants and risk polymorphisms in a few Hungarian SP-HUS patients, and have now extended these studies to a cohort of 13 Spanish SP-HUS patients. Five patients presented rare complement variants of unknown significance, but the frequency of the risk haplotypes in the CFH-CFHR3-CFHR1 region was similar to the observed in aHUS. Moreover, we observed desialylation of Factor H (FH) and the FH-Related proteins in plasma samples from 2 Spanish and 4 Hungarian SP-HUS patients. To analyze the functional relevance of this finding, we compared the ability of native and “in vitro” desialylated FH in: (a) binding to C3b-coated microtiter plates; (b) proteolysis of fluid-phase and surface-bound C3b by Factor I; (c) dissociation of surface bound-C3bBb convertase; (d) haemolytic assays on sheep erythrocytes. We found that desialylated FH had reduced capacity to control complement activation on sheep erythrocytes, suggesting a role for FH sialic acids on binding to cellular surfaces. We conclude that aHUS-risk variants in the CFH-CFHR3-CFHR1 region could also contribute to disease-predisposition to SP-HUS, and that transient desialylation of complement FH by the pneumococcal neuraminidase may have a role in disease pathogenesis.

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