[[(Arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives as high-affinity, selective 5-HT1A receptor ligands.

A series of 2-[[(4-aryl-1-piperazinyl)alkyl]thio]thieno[2,3-d]pyrimidin-4 (1H)-one and 3-substituted 2-[[(4-aryl-1-piperazinyl)alky]thio]thieno[2,3-d]pyrimidin-4 (3H)-one derivatives was prepared and evaluated for in vitro 5-HT1A receptor affinity by radioligand binding assays; the selectivity for 5-HT1A receptors rather than alpha 1-adrenoceptors was also examined (ratio of the IC50 alpha 1 to IC50 5-HT1A). The binding tests gave indications about the best features of the [(arylpiperazinyl)alkyl]thio moiety and of the substituents on the thiophene and pyrimidinone rings for efficacious and selective 5-HT1A ligands. The most effective derivative for displacing [3H]-8-OH-DPAT from rat hippocampal membranes was the 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl] propyl]thio]-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (70) (IC50 = 0.3 nM) with selectivity of 24 for the 5-HT1A over the alpha 1-adrenoceptor. Compound 73, where the 2-methoxyphenyl on the N4 piperazine ring was replaced with a pyrimidine group, showed the best selectivity, with a ratio of 74, while its affinity IC50 for 5-HT1A was 6.8 nM. These results, compared to those for compounds 46 (IC50 24 nM; selectivity 2) and 49 (IC50 226 nM; selectivity 5), N3 unsubstituted analogues of derivatives 70 and 73, show the importance of an amino group in position 3 of the thienopyrimidine system for the interaction with 5-HT1A receptor binding sites, although this fragment can affect the affinity and selectivity only if linked to the (arylpiperazinyl)alkyl moiety. The better selectivity of piperidine 74 (IC50 0.8; selectivity 45) compared to the analogous piperazine 70 is also noteworthy. Twenty of the 30 molecules used for determining the binding affinity to 5-HT1A and alpha 1-adrenergic receptors were selected for QSAR analysis using a series of molecular descriptors and calculated with the TSAR software.

High affinity and selectivity of [[(arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives for the 5-HT(1A) receptor. Synthesis and structure-affinity relationships.

In this work we report the affinity of new thienopyrimidinones for 5-HT(1A)Rs and the selectivity versus alpha(1)ARs. The 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-6-ethyl -thieno[2,3-d]pyrimidin-4(3H)-one 27 is the most potent and selective (Ki 0.19 nM, selectivity 115). Compound 31 with the N4 piperazine orthonitrophenyl nucleus instead of the orthomethoxyphenyl also shows a good affinity and selectivity (Ki 1. 46 nM, selectivity 84). The results of derivatives 28, 29 and 30 (Ki 3.28, 12.59 and 4.38 nM; selectivity 24, 4 and 5, respectively), which have, respectively, an ethyl, an allyl and an acetylamino group instead of an N3 amino group, indicate the importance of this last group for the interaction with 5-HT(1A)R. Comparison of the results for the superior homologue 53 (Ki 3.72 nM, selectivity 51) and the inferior homologue 52 (5-HT(1A) Ki 1499 nM, alpha(1)A Ki NA) of 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-6,7-dimethyl-8H-[1, 3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one 57 (Ki 23 nM, selectivity 5) shows how important the length of the chain binding the two heterocyclic systems is in the interaction with 5-HT(1A)Rs and alpha(1)ARs.

Manganese peroxidase from Phanerochaete chrysosporium. A homology-based molecular model.

A detailed three-dimensional model of manganese peroxidase was constructed using lignine peroxidase as the structural scaffold. This is the only protein in the peroxidase family except for cytochrome c peroxidase for which a resolved crystal structure is available. The model was built using the following procedure: (a) structurally preserved regions were derived from similar regions in the sequence alignment of the two proteins; (b) non-similar regions were modelled by searching a set of resolved protein structures for fragments which fitted in geometrically and choosing the best fitting fragment. Side chains were constructed by calculating rotamer-rotamer interaction energies and minimizing intramolecular energy. Model refinement was performed by molecular mechanics calculation. The quality of the model was assessed on the basis of the propensity of the amino acids to be inserted into regular secondary-structure elements and to be exposed to solvent. All the lignine peroxidase regions not used for model construction because of the lack of similarity, except the helix fragment Leu261-Phe269, correspond to external loops, suggesting reliable modelling. The manganese peroxidase model structure was analyzed in detail and several functionally relevant structural features were predicted, the most important being: (a) the very close structural similarity between lignine and manganese peroxidase active sites, suggesting a similar mode of hydrogen peroxide activation; (b) the substitution of polar residues for the hydrophobic amino acids exposed at the edge of the channel involved in substrate recognition in lignine peroxidase, suggesting that manganese peroxidase does not directly bind aromatic substrates; (c) the location of residues potentially able to bind Mn2+, spatially positioned on the side of the 3-CH3 heme edge.

Migration of natural killer cells across endothelial cell monolayers.

Under certain conditions, NK cells accumulate rapidly at extrahematic sites. In an effort to define the mechanisms underlying recruitment of NK cells in tissues, we investigated their ability to migrate across endothelial cell (EC) monolayers. A considerable proportion of NK cells adhered to EC and about 30 to 40% of the adherent NK cells migrated across EC. NK cells were two to three times more efficient than resting unseparated T cells but were considerably less efficient than monocytes. Exposure of NK cells to IL-2 or of EC to IL-1, TNF, or IFN-gamma augmented transendothelial migration. mAb directed against CD18 and CD11a inhibited binding and migration of NK cells across resting or IL-1-activated EC, whereas anti-CD11b and c mAb did not. Thus, the LFA-1 pathway is a crucial determinant of the adhesive and migratory interactions of NK cells with vascular endothelium. Using IL-1-activated EC, We found that anti-VLA-4 and anti-VCAM-1 mAb, utilized in concert with anti-CD18, significantly reduced adhesion and transmigration. The CS-1 peptide of fibronectin had no effect on binding and migration but, when used in concert with anti-CD18 and anti-VCAM-1 (but not anti-VLA-4), caused a small, but significant, increase in inhibition. The capacity to bind and migrate across endothelial monolayers underlies the recruitment of NK cells in tissues under certain physiologic and pathologic conditions.

The chloroperoxidase-catalyzed oxidation of phenols. Mechanism, selectivity, and characterization of enzyme-substrate complexes.

The reactivity of a series of para-substituted phenolic compounds in the peroxidation catalyzed by chloroperoxidase was investigated, and the results were interpreted on the basis of the binding characteristics of the substrates to the active site of the enzyme. Marked selectivity effects are observed. These operate through charge, preventing phenolic compounds carrying amino groups on the substituent chain to act as substrates for the enzyme, and through size, excluding potential substrates containing bulky substituents to the phenol nucleus. Also, chiral recognition is exhibited by chloroperoxidase in the oxidation of N-acetyltyrosine, where only the L isomer is oxidized. Kinetic measurements show that, in general, the efficiency of chloroperoxidase in the oxidation of phenols is lower than that of horseradish peroxidase. Paramagnetic NMR spectra and relaxation rate measurements of chloroperoxidase-phenol complexes are consistent with binding of the substrates close to the heme, in the distal pocket, with the phenol group pointing toward the iron atom. On the other hand, phenolic compounds which are not substrates for chloroperoxidase bind to the enzyme with a much different disposition, with the phenol group very distant from the iron and probably actually outside the active-site cavity.

Conformational polymorphism of the amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein.

Prion-related encephalopathies are characterized by cerebral accumulation of a post-translationally modified form of the cellular prion protein (PrPC), designated PrPSc. Evidence suggests that the conversion from PrPC to PrPSc involves changes in the secondary structure leading to an increase in beta-sheet content. We have previously shown that a synthetic peptide homologous to residues 106-126 of human PrP, belonging to a predicted alpha-helical domain, exhibits a beta-sheet conformation, forms amyloid-like fibrils, and is neurotoxic in vitro. The present study investigated how different chemicophysical conditions such as pH and ionic strength or a membrane-like environment influenced the secondary structure of this peptide. PrP 106-126 exhibited a predominantly beta-sheet structure in 200 mM phosphate buffer, pH 5.0, but a combination of beta-sheet and random coil structure in 200 mM phosphate buffer, pH 7.0, or in deionized water. The addition of trifluoroethanol (50% final concentration) to solutions of peptide in deionized water induced the appearance of an alpha-helical secondary structure, but did not modify the beta-sheet conformation of the peptide dissolved in 200 mM phosphate buffer, pH 5.0. In the presence of micelles formed by a 5% solution of sodium dodecyl sulfate, PrP 106-126 showed a high content of alpha-helix. When the peptide was dissolved in 5 mM phosphate buffer, pH 7.4, and incubated with liposomes, it changed from a prevalently random coil structure to a beta-sheet conformation. The environment-dependent conformational polymorphism of PrP 106-126 and its marked tendency to form stable beta-sheet structures at acidic pH could account for the shift from alpha-helix to beta-sheet associated with the conversion of PrPC to PrPSc, which occurs most likely in the endosomal-lysosomal compartment.

Molecular characteristics of a protease-resistant, amyloidogenic and neurotoxic peptide homologous to residues 106-126 of the prion protein.

In the prion-related encephalopathies the prion protein is converted to an altered form, known as PrPSc, that is partially resistant to protease digestion. This abnormal isoform accumulates in the brain and its protease-resistant core aggregates extracellularly into amyloid fibrils. We have investigated the conformational properties, aggregation behaviour and sensitivity to protease digestion of a synthetic peptide homologous to residues 106-126 of human PrP, which was previously found to form amyloid-like fibrils in vitro and displayed neurotoxic activity toward primary cultures of rat hippocampal neurons. A scrambled sequence of peptide PrP 106-126 was used as a control. By circular dichroism, PrP 106-126 exhibited a secondary structure composed largely of beta-sheet, whereas the scrambled sequence of PrP 106-126 showed a random coil structure. The beta-sheet content of PrP 106-126 was much higher in 200 mM phosphate buffer at pH 5.0 than in the same buffer at pH 7.0. Laser light scattering analysis showed that PrP 106-126 aggregated immediately after dissolution in 20 mM or 200 mM phosphate buffer, pH 5.0 and 7.0, whereas scrambled PrP 106-126 did not. PrP 106-126 aggregates had an average hydrodinamic diameter of 100 nm and an average molecular weight of 12 x 10(6) +/- 30% Daltons, corresponding to the aggregation of 6000 +/- 30% molecules. Peptide PrP 106-126 showed partial resistance to digestion with Proteinase K and Pronase, whereas scrambled PrP 106-126 was completely degraded by incubation with the enzymes at 37 degrees C for 30 minutes.

beta PP and Tau interaction. A possible link between amyloid and neurofibrillary tangles in Alzheimer's disease.

Extracellular deposition of amyloid fibrils and intraneuronal accumulation of paired helical filaments (PHFs) are the neuropathological hallmarks of Alzheimer's disease. The major constituent of amyloid fibrils is a 39- to 43-residue peptide (termed A beta), which is derived from a 695- to 770-amino-acid precursor protein (termed beta PP). The main component of PHFs identified so far is the microtubule-associated protein tau. Yet, there is no direct evidence of interconnection between these two pathological states. We report here that antibodies to an epitope located between residues 713 and 723 of beta PP770 (ie, the transmembrane region of beta PP distal to A beta) consistently labeled PHFs in the brain of Alzheimer patients. Solid phase immunoassay showed that a peptide homologous to residues 713 to 730 of beta PP770 bound tau proteins. This beta PP peptide spontaneously formed fibrils in vitro and, in the presence of tau, generated dense fibrillary assemblies containing both molecules. These data suggest that beta PP or beta PP fragments containing the tau binding site are involved in the pathogenesis of PHFs in Alzheimer's disease.