Identification and biological evaluation of a series of 1H-benzo[de]isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B polymerase inhibitors.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new series of selective inhibitors of HCV NS5B polymerase.

Structural basis for resistance of the genotype 2b hepatitis C virus NS5B polymerase to site A non-nucleoside inhibitors.

Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Lambda1 loop in the apoenzyme.

Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization.

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography.

Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine produces a dramatic rearrangement in the active-site gorge.

The X-ray crystal structures were solved for complexes with Torpedo californica acetylcholinesterase of two bivalent tacrine derivative compounds in which the two tacrine rings were separated by 5- and 7-carbon spacers. The derivative with the 7-carbon spacer spans the length of the active-site gorge, making sandwich interactions with aromatic residues both in the catalytic anionic site (Trp84 and Phe330) at the bottom of the gorge and at the peripheral anionic site near its mouth (Tyr70 and Trp279). The derivative with the 5-carbon spacer interacts in a similar manner at the bottom of the gorge, but the shorter tether precludes a sandwich interaction at the peripheral anionic site.

FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded.

An easy-to-use, versatile and freely available graphic web server, FoldIndex is described: it predicts if a given protein sequence is intrinsically unfolded implementing the algorithm of Uversky and co-workers, which is based on the average residue hydrophobicity and net charge of the sequence. FoldIndex has an error rate comparable to that of more sophisticated fold prediction methods. Sliding windows permit identification of large regions within a protein that possess folding propensities different from those of the whole protein.

The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded.

Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size.

Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids.

The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes.