Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive, whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line. The model has been validated by demonstrating that both a small molecule NS3/4A protease inhibitor (BILN 2061) and human interferon- alpha (IFN-alpha) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. The efficacy of protease inhibitor plus IFN-alpha demonstrated the application of the model for testing compounds in combination therapies. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo.
Methods Mol Biol 2009
PMID:A hepatitis C virus xenograft mouse efficacy model. 1934 4

Dengue virus belongs to the family Flaviviridae and is a major emerging pathogen for which the development of vaccines and antiviral therapy has seen little success. The NS3 viral protease is a potential target for antiviral drugs since it is required for virus replication. The goal of this study was to identify novel dengue virus (type 2; DEN2V) protease inhibitors for eventual development as effective anti-flaviviral drugs. The EUDOC docking program was used to computationally screen a small-molecule library for compounds that dock into the P1 pocket and the catalytic site of the DEN2V NS3 protease domain apo-structure [Murthy, K., Clum, S., Padmanabhan, R., 1999. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J. Biol. Chem. 274, 5573-5580] and the Bowman-Birk inhibitor-bound structure [Murthy, K., Judge, K., DeLucas, L., Padmanabhan, R., 2000. Crystal structure of dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. J. Mol. Biol. 301, 759-767]. The top 20 computer-identified hits that demonstrated the most favorable scoring "energies" were selected for in vitro assessment of protease inhibition. Preliminary protease activity assays demonstrated that more than half of the tested compounds were soluble and exhibited in vitro inhibition of the DEN2V protease. Two of these compounds also inhibited viral replication in cell culture experiments, and thus are promising compounds for further development.
 ...
PMID:Structure-based discovery of dengue virus protease inhibitors. 1942 1

The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs.We established a genetic screen that is based on coexpression of NS3, a beta-galactosidase reporter that is cleavable by NS3, and potential inhibitors within the same bacterial cell. A single-chain antibody (scFv) library was prepared from spleens of NS3-immunized mice and the screen was used to isolate a panel of protease-inhibiting scFvs. Candidate scFvs were validated for inhibitory activity using an o-nitrophenyl-beta-galactoside (ONPG) hydrolysis assay.The methods can be used more generally to isolate protease-inhibiting cytoplasmic intrabodies able to inhibit proteases or other activities that can be linked with the phenotype of Escherichia coli.
Methods Mol Biol 2009
PMID:Isolation of scFvs that inhibit the NS3 protease of hepatitis C virus by a combination of phage display and a bacterial genetic screen. 1955 91

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor-complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz-Nle(P4)-Lys(P3)-Arg(P2)-Arg(P1)-H) were generated. The first DV protease model (DV-1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV-2) was built taking the WNV NS3/NS2B protease in the inhibitor-complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure-based design purposes.
J Mol Recognit
PMID:Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction. 1969 93

The spatial dynamics of the West Nile Virus epidemic in North America are largely unknown. Previous studies that investigated the evolutionary history of the virus used sequence data from the structural genes (prM and E); however, these regions may lack phylogenetic information and obscure true evolutionary relationships. This study systematically evaluated the evolutionary patterns in the eleven genes of the WNV genome in order to determine which region(s) were most phylogenetically informative. We found that while the E region lacks resolution and can potentially result in misleading conclusions, the full NS3 or NS5 regions have strong phylogenetic signal. Furthermore, we show that geographic structure of WNV infection within the US is more pronounced than previously reported in studies that used the structural genes. We conclude that future evolutionary studies should focus on NS3 and NS5 in order to maximize the available sequences while retaining maximal interpretative power to infer temporal and geographic trends among WNV strains.
Mol Phylogenet Evol 2010 Jul
PMID:Evolutionary characterization of the West Nile Virus complete genome. 2010 43

The development of techniques based on fluorescence has made it possible to create new types of assays that represent an advantageous alternative to old tests relying on radioactivity. Such a novel approach has been applied to develop a high-throughput assay to measure the helicase activity of the hepatitis C virus (HCV) NS3 protein and the inhibitory potential of several classes of compounds. The NS3 helicase is one of the most promising targets of anti-HCV-oriented screening of compounds due to the urgent need for more effective and tolerable drugs. The 96- or 384-well microplate assay that we developed is based on the use of a quenched double-stranded DNA substrate labeled with a fluorophore (Cy3 or FAM) and with a Black Hole Quencher 1 or 2. It allows for direct (real-time) measurements of substrate unwinding and inhibition of unwinding by anti-helicase compounds. After a few modifications of buffers and assay conditions this method can be applied to various variants of HCV helicase and other proteins with helicase activities.
Methods Mol Biol 2010
PMID:Fluorometric assay of hepatitis C virus NS3 helicase activity. 2022 52

The hepatitis C virus NS3 protein contains an N-terminal serine protease and a C-terminal helicase that unwinds RNA or DNA duplexes. The HCV NS3 protein is the target for several antiviral drugs in clinical trials, which inhibit the protease function. A method is reported to simultaneously monitor the helicase and protease function of the NS3 protein in a single reaction using fluorescence spectroscopy and a single chain recombinant protein where NS3 is fused to its protease activator NS4A. The method monitors both activities together in real time and is amenable to high-throughput screening. This new procedure could be used to identify compounds that inhibit both the helicase and protease activity of NS3.
Methods Mol Biol 2010
PMID:A method to simultaneously monitor hepatitis C virus NS3 helicase and protease activities. 2022 53

In this chapter, we will demonstrate the usage of a suite of computational techniques based on a coarse-grained elastic network model by applying them to a monomeric helicase-the NS3 helicase of hepatitis C virus. These techniques allow us to predict and visualize collective domain motions encoded in the protein structures, probe allosteric couplings between key functional sites (such as ATP-binding site and DNA/RNA-binding site of a helicase), and simulate ATP-binding-induced global conformational changes. These general techniques are not only applicable to NS3 but also to other multi-domain protein structures.
Methods Mol Biol 2010
PMID:Computer modeling of helicases using elastic network model. 2022 54

Serine protease activity of the NS3 protein of Dengue virus is an important target of antiviral agents that interfere with the viral polyprotein precursor processing catalyzed by the NS3 protease (NS3pro), which is important for the viral replication and maturation. Recent studies showed that substrate-based peptidomimetics carrying an electrophilic warhead inhibit the NS2B-NS3pro cofactor-protease complex with inhibition constants in the low micromolar concentration range when basic amino acid residues occupy P(1) and P(2) positions of the inhibitor, and an aldehyde warhead is attached to the P(1). We have used computer-assisted combinatorial techniques to design, focus using the NS2B-NS3pro receptor 3D structure, and in silico screen a virtual library of more than 9,200 peptidomimetic analogs targeted around the template inhibitor Bz-Nle-Lys-Arg-Arg-H (Bz-benzoyl) that are composed mainly of unusual amino acid residues in all positions P(1)-P(4). The most promising virtual hits were analyzed in terms of computed enzyme-inhibitor interactions and Adsorption, Distribution, Metabolism and Excretion (ADME) related physico-chemical properties. Our study can direct the interest of medicinal chemists working on a next generation of antiviral chemotherapeutics against the Dengue Fever towards the explored subset of the chemical space that is predicted to contain peptide aldehydes with NS3pro inhibition potencies in nanomolar range which display ADME-related properties comparable to the training set inhibitors.
J Comput Aided Mol Des 2010 Mar
PMID:Design, structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease. 2030 83

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long alpha-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5'-triphosphate, cytidine 5'-triphosphate, and uridine 5'-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.
J Mol Biol 2010 Jul 23
PMID:Crystal structure of human RNA helicase A (DHX9): structural basis for unselective nucleotide base binding in a DEAD-box variant protein. 2051 Feb 46


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>