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1.
Compr Physiol ; 7(4): 1449-1461, 2017 Sep 12.
Article in English | MEDLINE | ID: mdl-28915331

ABSTRACT

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.


Subject(s)
Dual-Specificity Phosphatases/metabolism , Enzyme Inhibitors/pharmacology , Molecular Docking Simulation , Animals , Binding Sites , Dual-Specificity Phosphatases/antagonists & inhibitors , Dual-Specificity Phosphatases/chemistry , Dual-Specificity Phosphatases/genetics , Enzyme Inhibitors/chemistry , Humans , MAP Kinase Signaling System , Protein Binding
2.
Antibiotics (Basel) ; 6(1)2017 Jan 28.
Article in English | MEDLINE | ID: mdl-28134858

ABSTRACT

The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics.

3.
ACS Infect Dis ; 1(3): 140-148, 2015 Mar 13.
Article in English | MEDLINE | ID: mdl-26029739

ABSTRACT

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 µM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.

4.
ACS Chem Biol ; 10(8): 1887-96, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-25961497

ABSTRACT

This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevir-resistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir and boceprevir, minor synergy was observed with danoprevir, and modest synergy was observed with grazoprevir.


Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Hepacivirus/chemistry , Hepacivirus/metabolism , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Models, Molecular , Molecular Targeted Therapy , Oligopeptides/pharmacology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
5.
Eur J Med Chem ; 96: 250-8, 2015.
Article in English | MEDLINE | ID: mdl-25890075

ABSTRACT

Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC50 values of 7.9 and 2.6 µM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.


Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Hepacivirus/drug effects , Indoles/pharmacology , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Microbial Sensitivity Tests , Structure-Activity Relationship
6.
ACS Chem Biol ; 9(10): 2393-403, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25126694

ABSTRACT

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 µM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 µM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.


Subject(s)
Antiviral Agents/pharmacology , Azoles/pharmacology , Hepatitis C/virology , Nucleic Acids/metabolism , Organoselenium Compounds/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Antioxidants/pharmacology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/virology , Cell Proliferation , Electrophoretic Mobility Shift Assay , Hepacivirus/drug effects , Hepatitis C/drug therapy , Hepatitis C/metabolism , Humans , Hydrolysis , Isoindoles , Kinetics , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/virology , Models, Molecular , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , Tumor Cells, Cultured
7.
Biochemistry ; 52(36): 6151-9, 2013 Sep 10.
Article in English | MEDLINE | ID: mdl-23947785

ABSTRACT

Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) and reveals that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.


Subject(s)
Adenosine Triphosphate/metabolism , Aurintricarboxylic Acid/pharmacology , DNA/metabolism , Viral Nonstructural Proteins/drug effects , Amino Acid Substitution , Calorimetry, Differential Scanning , Hepacivirus/enzymology , Models, Molecular , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
8.
J Biol Chem ; 288(27): 19949-57, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23703611

ABSTRACT

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 µM. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.


Subject(s)
Adenosine Triphosphate/chemistry , Hepacivirus/enzymology , Models, Molecular , RNA Helicases/chemistry , RNA, Viral , Thiazoles/chemistry , Viral Proteins/chemistry , Adenosine Triphosphate/metabolism , Hepacivirus/genetics , Hydrolysis , Mutagenesis, Site-Directed , RNA Helicases/genetics , RNA Helicases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
9.
Antiviral Res ; 96(2): 245-55, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22940425

ABSTRACT

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.


Subject(s)
Antiviral Agents/pharmacology , DNA Helicases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Serine Endopeptidases/metabolism , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Cell Line , Fluorescence , Hepacivirus/drug effects , Humans , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Protein Binding , Thiazoles/chemistry
10.
Nucleic Acids Res ; 40(17): 8607-21, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22740655

ABSTRACT

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50=1.4 µM), suramin sodium salt (IC50=3.6 µM), NF 023 hydrate (IC50=6.2 µM) and tyrphostin AG 538 (IC50=3.6 µM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.


Subject(s)
Enzyme Inhibitors/pharmacology , Hepacivirus/enzymology , High-Throughput Screening Assays , RNA Helicases/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , DNA/metabolism , DNA-Binding Proteins/metabolism , Enzyme Assays , Escherichia coli Proteins/metabolism , Fluorescence Polarization , RNA Helicases/metabolism , Small Molecule Libraries , Viral Nonstructural Proteins/metabolism
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