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1.
Antiviral Res ; 147: 149-158, 2017 Nov.
Article in English | MEDLINE | ID: mdl-29037976

ABSTRACT

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Drug Resistance, Viral/genetics , Viral Nonstructural Proteins/genetics , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Antiviral Agents/toxicity , Cell Line, Tumor , Chlorocebus aethiops , Dengue , Dengue Virus/genetics , Dengue Virus/physiology , Drug Discovery , Drug Resistance, Viral/drug effects , Hepacivirus/genetics , Humans , Mutation , RNA, Viral/genetics , Replicon/drug effects , Sequence Analysis, RNA , Small Molecule Libraries , Vero Cells
2.
Methods Mol Biol ; 1030: 185-94, 2013.
Article in English | MEDLINE | ID: mdl-23821269

ABSTRACT

Dengue, a mosquito-borne virus of the Flaviviridae family, is reemerging as one of the most important human pathogens in tropical and subtropical regions of the world. It is estimated that 2.5 billion people live in areas at risk for transmission of dengue virus (DENV). Furthermore, it causes significant morbidity and mortality with 50-100 million infections per year. Currently, there are no vaccines commercially available and no effective antiviral drugs for treatment of DENV infections. In this chapter, we describe a plaque reduction assay and a cell-based high-throughput antiviral assay for identifying inhibitors against DENV. The latter is a homogeneous high-throughput assay that has been fully validated according to the Food and Drug Administration (FDA) guidelines for assay validation and can be used for screening compound libraries.


Subject(s)
Antiviral Agents/pharmacology , Cell Culture Techniques , Dengue Virus/drug effects , High-Throughput Screening Assays , Microbial Sensitivity Tests/methods , Animals , Antiviral Agents/toxicity , Cell Line , Chlorocebus aethiops , Cricetinae , Humans , Vero Cells
3.
Methods Mol Biol ; 1030: 195-203, 2013.
Article in English | MEDLINE | ID: mdl-23821270

ABSTRACT

We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, ß-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells ß-actin gene is 100%.


Subject(s)
Antiviral Agents/pharmacology , Dengue/drug therapy , Dengue/genetics , Microbial Sensitivity Tests/methods , Reverse Transcriptase Polymerase Chain Reaction , Animals , Chlorocebus aethiops , Dengue/classification , Humans , Inhibitory Concentration 50 , Reverse Transcriptase Polymerase Chain Reaction/methods , Serotyping , Vero Cells
4.
Methods Mol Biol ; 1030: 237-47, 2013.
Article in English | MEDLINE | ID: mdl-23821273

ABSTRACT

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.


Subject(s)
Antiviral Agents/pharmacology , Dengue Virus/drug effects , Dengue Virus/physiology , Gene Expression , High-Throughput Screening Assays , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Enzyme Assays/methods , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Plasmids/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transformation, Bacterial , Viral Nonstructural Proteins/isolation & purification
6.
Bioorg Med Chem Lett ; 19(9): 2492-6, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19342234

ABSTRACT

Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.


Subject(s)
Antiviral Agents/chemical synthesis , Benzodiazepines/chemistry , Chemistry, Pharmaceutical/methods , Hepacivirus/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Acrylates/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Drug Design , Hepacivirus/enzymology , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Structure , Structure-Activity Relationship
7.
Front Biosci (Landmark Ed) ; 14(8): 2904-10, 2009 01 01.
Article in English | MEDLINE | ID: mdl-19273243

ABSTRACT

Pancreatic cancer is one of the deadliest diseases largely due to difficulty in early diagnosis and the lack of effective treatments. KRAS is mutated in more than 90% of pancreatic cancer patients, and oncogenic KRAS contributes to pancreatic cancer tumorigenesis and progression. In this report, using an oncogenic KRASV12-based pancreatic cancer cell model, we developed a chemical genetic screen to identify small chemical inhibitors that selectively target pancreatic cancer cells with gain-of-function KRAS mutation. After screening ~3,200 compounds, we identified one compound that showed selective synthetic lethality against the KRASV12 transformed human pancreatic ductal epithelial cell over its isogenic parental cell line. These selective KRASV12-synthetic lethal compounds may serve as leads for subsequent development of clinically-effective treatments for pancreatic cancer.


Subject(s)
Antineoplastic Agents/therapeutic use , Genes, ras , Pancreatic Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Cell Line , Cell Transformation, Neoplastic , Drug Screening Assays, Antitumor , Humans , Pancreatic Neoplasms/genetics
8.
Bioorg Med Chem Lett ; 18(1): 427-31, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17981463

ABSTRACT

This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasminogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were identified that are viable substrates for the protease.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Peptides/chemical synthesis , Peptides/pharmacology , Plasminogen Activators/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Fluorometry , Kinetics , Molecular Sequence Data , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides/chemistry , Plasminogen Activators/chemistry , Plasminogen Activators/metabolism , Protease Inhibitors/chemistry , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Yersinia pestis/enzymology
9.
Bioorg Med Chem Lett ; 17(21): 5940-3, 2007 Nov 01.
Article in English | MEDLINE | ID: mdl-17826998

ABSTRACT

This manuscript describes methods appropriate for the parallel synthesis of libraries based on the tricyclic thioxanthen-9-one-10,10-dioxide scaffold. The novel compounds were synthesized from previously reported 3-chlorothioxanthen-9-one-10,10-dioxide and commercially available 3-carboxylic acid thioxanthen-9-one-10,10-dioxide. The library members were screened for activity in a fluorescence polarization assay for inhibitors of BRCT domains of breast cancer gene 1 and in cell-based secreted alkaline phosphatase reported replicon system for activity against hepatitis C virus.


Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Xanthenes/chemical synthesis , Xanthenes/pharmacology , Fluorescence Polarization , Hepacivirus/drug effects
10.
Org Lett ; 9(18): 3507-10, 2007 Aug 30.
Article in English | MEDLINE | ID: mdl-17676858

ABSTRACT

Lewis acid-mediated intramolecular attack of vinylsilanes at tethered oxonium precursors 1 results in a rapid assembly of the cis-fused bicyclic ether species 3, with complete regio- and stereospecific control, and in some cases near-quantitative yield. Continued investigation suggests a general approach to a variety of such ether frameworks.


Subject(s)
Bridged Bicyclo Compounds/chemical synthesis , Ethers, Cyclic/chemical synthesis , Silanes/chemistry , Vinyl Compounds/chemistry , Bridged Bicyclo Compounds/chemistry , Cyclization , Ethers, Cyclic/chemistry , Molecular Structure , Oxygen/chemistry , Stereoisomerism
11.
Org Biomol Chem ; 4(16): 3155-65, 2006 Aug 21.
Article in English | MEDLINE | ID: mdl-16886085

ABSTRACT

N-Alkylation of 4,5-dihydroimidazoles with alkene-containing bromomethyl ketones and treatment of the so-formed 4,5-dihydroimidazolium ions with DBU gives rise to an intramolecular 1,3-dipolar cycloaddition reaction that affords (via a reaction cascade involving eliminative ring-opening, recyclisation and prototropic tautomerism) unexpected hexahydropyrrolo[1,2,3-de]quinoxaline products. Steric bulk in both the dihydroimidazole and the dipolarophile allows isolation of an imidazo[1,2-a]indole, the initial product of cycloaddition. When the bromomethyl ketone contains no other functionality, or when cycloaddition is inhibited due to steric constraints, the dihydroimidazolium ion undergoes ring-opening hydrolysis followed by recyclization of the exposed amino ketone to afford either 3-alkyl-1-formylpiperazine-2-ones or 3-aryl-1-formyl-1,4,5,6-tetrahydropyrazines.

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