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1.
J Virol ; 89(10): 5362-70, 2015 May.
Article in English | MEDLINE | ID: mdl-25740995

ABSTRACT

UNLABELLED: Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing. IMPORTANCE: The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of protease activity can block RNA replication by preventing expression of mature replicase components. Like many viral proteins, NS3 is multifunctional, but how PIs affect stages of the HCV life cycle beyond polyprotein processing has not been well studied. Using cell-based assays, we show here that PIs can directly inhibit viral RNA synthesis and also block a late stage in virus assembly/maturation at clinically relevant concentrations.


Subject(s)
Carrier Proteins/antagonists & inhibitors , Carrier Proteins/physiology , Hepacivirus/drug effects , Hepacivirus/physiology , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/physiology , Virus Assembly/drug effects , Virus Assembly/physiology , Antiviral Agents/pharmacology , Cell Line , Humans , Intracellular Signaling Peptides and Proteins , Kinetics , Oligopeptides/pharmacology , Protein Processing, Post-Translational/drug effects , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/metabolism
2.
ChemMedChem ; 9(4): 823-32, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24616449

ABSTRACT

Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,ß-methylene adenosine 5'-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.


Subject(s)
Adenylyl Cyclase Inhibitors , Bicarbonates/pharmacology , Enzyme Inhibitors/pharmacology , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/metabolism , Bicarbonates/chemical synthesis , Bicarbonates/chemistry , Catalytic Domain/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Structure-Activity Relationship
3.
Nat Chem Biol ; 8(11): 920-5, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23023261

ABSTRACT

Here we report a highly conserved new binding site located at the interface between the protease and helicase domains of the hepatitis C virus (HCV) NS3 protein. Using a chemical lead, identified by fragment screening and structure-guided design, we demonstrate that this site has a regulatory function on the protease activity via an allosteric mechanism. We propose that compounds binding at this allosteric site inhibit the function of the NS3 protein by stabilizing an inactive conformation and thus represent a new class of direct-acting antiviral agents.


Subject(s)
Allosteric Site , Viral Nonstructural Proteins/metabolism , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Allosteric Site/genetics , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Dose-Response Relationship, Drug , Ligands , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Viral Nonstructural Proteins/drug effects , Viral Nonstructural Proteins/genetics
4.
Mol Cancer Ther ; 10(9): 1542-52, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21764904

ABSTRACT

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.


Subject(s)
Antineoplastic Agents/pharmacology , Fibroblast Growth Factors/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Design , Drug Evaluation, Preclinical , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Receptors, Fibroblast Growth Factor/genetics , Signal Transduction/drug effects , Treatment Outcome , Xenograft Model Antitumor Assays
5.
J Mol Biol ; 367(3): 882-94, 2007 Mar 30.
Article in English | MEDLINE | ID: mdl-17275837

ABSTRACT

Although the crystal structure of the anti-cancer target protein kinase B (PKBbeta/Akt-2) has been useful in guiding inhibitor design, the closely related kinase PKA has generally been used as a structural mimic due to its facile crystallization with a range of ligands. The use of PKB-inhibitor crystallography would bring important benefits, including a more rigorous understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a "back-soaking" method for obtaining PKBbeta-ligand crystal structures, and provide a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and the compound adopts a similar binding mode in all three systems. By contrast, the PKB-selective inhibitor A-443654 adopts a conformation in PKB and PKA-PKB that differs from that with PKA. We provide a structural explanation for this difference, and highlight the ability of PKA-PKB to mimic the true PKB binding mode in this case.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/chemistry , Animals , Binding Sites , Cattle , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , In Vitro Techniques , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Static Electricity
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