Factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of neutral P1 substituents

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for neutral ligands, which bind in the S1 pocket of factor Xa were investigated with the 2-carboxyindole scaffold. This privileged fragment assembly approach yielded a set of equipotent, selective inhibitors with structurally diverse neutral P1 substituents.     

Novel bisbenzamidines as potential drug candidates for the treatment of Pneumocystis carinii pneumonia

A series of pentamidine congeners has been synthesized and screened for their in vitro activity against Pneumocystis carinii. Among the tested compounds, bisbenzamidines linked by a flexible pentanediamide or hexanediamide chain (7 and 9) emerged as exceptionally potent agents that were more effective and less toxic than pentamidine in the assays described in this study.     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Affinity parameters of amino acid derivative binding to molecularly imprinted nanospheres consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)

The binding of L-Boc-phenylalanine anilide (BFA) and L-Boc-phenylalanine (phe) to molecularly imprinted and non-imprinted polymer nanoparticles consisting of poly[(ethylene glycol dimethacrylate)-co-(methacrylic acid)] has been investigated by adsorption experiments and mathematical modeling. The experimental isotherms have been mathematically adapted following the models of Freundlich, Langmuir, Langmuir-Freundlich, Bi-Langmuir, and extended Langmuir. The extended Langmuir model differentiated between specific and nonspecific binding of the ligand to the receptor nanoparticles and rendered excellent fitting of the experimental data. It delivered a thermodynamic and kinetic parameter set on the experimental association curves of L-BFA by L-BFA-imprinted nanospheres in suspension experiments with the equilibrium constant KD= 4.09 +/- 0.69 micromol L(-1) and the kinetic association rate constant Ka= 5.60 mL micromol(-1) min(-1).     

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.     

Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification

Exostosin1 (Ext1) belongs to a family of glycosyltransferases necessary for the synthesis of the heparan sulfate (HS) chains of proteoglycans, which regulate signaling of several growth factors. Loss of tout velu (ttv), the homolog of Ext1 in Drosophila, inhibits Hedgehog movement. In contrast, we show that reduced HS synthesis in mice carrying a hypomorphic mutation in Ext1 results in an elevated range of Indian hedgehog (Ihh) signaling during embryonic chondrocyte differentiation. Our data suggest a dual function for HS: First, HS is necessary to bind Hedgehog in the extracellular space. Second, HS negatively regulates the range of Hedgehog signaling in a concentration-dependent manner. Additionally, our data indicate that Ihh acts as a long-range morphogen, directly activating the expression of parathyroid hormone-like hormone. Finally, we propose that the development of exostoses in the human Hereditary Multiple Exostoses syndrome can be attributed to activation of Ihh signaling.     

The sensitive giant: the role of titin-based stretch sensing complexes in the heart

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.     

Comparative genomics of Pneumocystis carinii with other protists: implications for life style

Three protistan genomes were analyzed for differential genetic traits that may be associated with biological adaptations to their unique life styles. The microsporidian, Encephalitozoon cuniculi, an obligate intracellular parasite; the ascomycetes, Pneumocystis carinii, considered an opportunistic pathogen; and Saccharomyces cerevisiae, a model organism exhibiting a free-living life style, were used in comparisons of genomic architecture, reproductive strategies, and metabolic capacity predicted by the presence of signature genes. Genome size, gene number, and metabolic function decreased as the organisms became more dependent on their hosts. In contrast, gene density and the percentage of genes dedicated to cell growth and division were substantially increased in the genome of E. cuniculi. The obligate life style was associated with reductions in gene number, genome size, and reduced metabolic capacity while the free-living life style was coincident with gene duplications and duplication of large portions of the genome. The genomic characteristics and metabolic capacity of P. carinii were usually intermediate between those of the other two protistan genomes, but unique characteristics such as the presence of a single rDNA locus may indicate that these organisms could be in the process of becoming more host dependent.