ABSTRACT
Four serotypes of dengue virus (DENV1-4), mosquito-borne members of Flaviviridae family cause frequent epidemics causing considerable morbidity and mortality in humans throughout tropical regions of the world. There is no vaccine or antiviral therapeutics available for human use. In a previous study, we reported that compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of West Nile virus serine protease. In this study, we analyzed potencies of some compounds with (8-HQ)-aminobenzothiazole derivatives for inhibition of DENV2 protease in vitro. We identified analogs 1-4 with 2-aminothiazole or 2-aminobenzothiazole scaffold with sub-micromolar potencies (IC(50)) in the in vitro protease assays. The kinetic constant (K(i)) for the most potent 8-HQ-aminobenzothiazole inhibitor (compound 1) with an IC(50) value of 0.91±0.05µM was determined to be 2.36±0.13µM. This compound inhibits the DENV2 NS2B/NS3pro by a competitive mode of inhibition.
Subject(s)
Benzothiazoles/pharmacology , Dengue Virus/enzymology , Oxyquinoline/pharmacokinetics , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Benzothiazoles/chemistry , Inhibitory Concentration 50 , Kinetics , Oxyquinoline/analogs & derivatives , Protease Inhibitors/chemistryABSTRACT
The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.
Subject(s)
RNA, Ribosomal/chemistry , Ribonucleoproteins/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Thermus thermophilus/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Pairing , Base Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , Ribonucleoproteins/metabolism , Ribosomal Protein S6 , Ribosomal Proteins/metabolism , Thermus thermophilus/ultrastructureABSTRACT
NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.
