Bookmark and Share
BioAssay: AID 623968

Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): RT-PCR-based cell-based assay to determine the effect of compounds on RNA levels

Name: Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): RT-PCR-based cell-based assay to determine the effect of compounds on RNA levels ..more
_
   
 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 623968
Data Source: The Scripps Research Institute Molecular Screening Center (HCV NS3_INH_RTPCR_1XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-04-02
Hold-until Date: 2013-03-30
Modify Date: 2013-03-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
Show more
AIDNameTypeProbeComment
1800Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screening depositor-specified cross reference: Primary screen (HCV NS3 helicase inhibitors in singlicate)
1830Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Summary depositor-specified cross reference: Summary (HCV NS3 helicase inhibitors)
1845Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors: biochemical high-throughput screening assay to identify compounds that cause fluorescent intercalator displacement (FID)Screening depositor-specified cross reference: Counterscreen (Fluorescent intercalator displacement in singlicate)
1943Fluorescence-based confirmation biochemical high throughput screening assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3)Screening depositor-specified cross reference: Confirmation screen (HCV NS3 helicase inhibitors in triplicate)
1945Fluorescence-based counterscreen assay for HCV NS3 helicase inhibitors: biochemical high-throughput screening assay to identify compounds that cause fluorescent intercalator displacement (FID) in triplicate.Screening depositor-specified cross reference: Counterscreen (Fluorescent intercalator displacement in triplicate)
2172Counterscreen for HCV NS3 helicase inhibitors: Fluorescence-based biochemical high-throughput dose response assay for compounds that cause fluorescent intercalator displacement (FID).Confirmatory depositor-specified cross reference: Dose response counterscreen (Fluorescent intercalator displacement in triplicate)
2173Fluorescence-based biochemical high throughput dose response assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).Confirmatory depositor-specified cross reference: Dose response (HCV NS3 helicase inhibitors in triplicate)
2474Late stage results for the probe development effort to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for inhibitors of NS3Confirmatory depositor-specified cross reference: Late stage dose response (HCV NS3 helicase inhibitors in triplicate)
2476Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): fluorescence-based biochemical dose response assay for compounds that cause fluorescent intercalator displacement (FID)Confirmatory depositor-specified cross reference: Late stage dose response counterscreen (Fluorescent intercalator displacement in triplicate )
463231Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxicOther depositor-specified cross reference: Late stage dose response counterscreen (Cytotoxicity)
463235Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds inhibit replication of HCV RNA repliconConfirmatory depositor-specified cross reference: Late stage dose response (Replication of HCV RNA replicon inhibitors)
485301Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strainsConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (Helicase encoded by HCV strains inhibitors)
504419Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 2Other depositor-specified cross reference: Late stage dose response counterscreen (Helicase encoded by HCV strains inhibitors)
588360Late-stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based dose response assay to determine whether compounds that inhibit replication of HCV RNA replicon are cytotoxicConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (Cytotoxicity)
602275Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): real-time florescence-based biochemical assay to determine whether compounds inhibit the helicase encoded by one or more HCV strains: Set 3Other1 same project related to Summary assay
623961Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based assay to determine cytotoxicity of compoundsOther same project related to Summary assay
623962Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): luminescence-based cell-based assay to determine whether compounds inhibit HCV replicationOther same project related to Summary assay
623964Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay to determine whether compounds inhibit the HCV protease NS3-NS4AConfirmatory same project related to Summary assay
623966Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence polarization-based biochemical assay to determine whether compounds can displace the E. coli single stranded DNA binding proteinConfirmatory same project related to Summary assay
623970Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay with SYBR to determine whether compounds bind DNAConfirmatory same project related to Summary assay
623972Late stage probe development counterscreen for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): florescence-based biochemical assay with EtBr to determine whether compounds bind DNAConfirmatory same project related to Summary assay
623973Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): absorbance-based biochemical assay to determine whether compounds inhibit ATPase activityConfirmatory same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frick, New York Medical College
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085690-01
Grant Proposal PI: David Frick, New York Medical College
External Assay ID: HCV NS3_INH_RTPCR_1XIC50

Name: Late stage probe development assay for inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3): RT-PCR-based cell-based assay to determine the effect of compounds on RNA levels

Description:

The flavivirus Hepatitis C Virus (HCV) is a major cause of liver failure and hepatocellular cancer, with about 170 million people infected worldwide (1). The HCV has a small RNA genome that is directly translated by the infected host cell into a single precursor polyprotein that is processed by enzymatic cleavage into 10 proteins of diverse function. The non-structural proteins include p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, and are responsible for the replication and packaging of the HCV genome into capsids formed by the structural proteins (core, E1, E2)(2). Replication of HCV in human cells requires the action of the HCV non-structural protein 3 (NS3). This enzyme exhibits dual NTPase/helicase activities and functions to unwind DNA/DNA, RNA/RNA, and RNA/DNA duplexes by disrupting hydrogen bonds that hold the two strands together (3). The HCV NS3 helicase mediates the "active" form of duplex unwinding, and thus is dependent upon NTP and at least two nucleic acid binding sites on the NS3 surface (3). HCV NS3 is able to target homotypic and heterotypic duplexes because the interaction between the enzyme and the DNA or RNA substrate is mediated by phosphate groups and not by the nucleotide base or sugar moieties (4). The current absence of a vaccine to prevent HCV infection (5), along with knockout studies showing that the helicase and/or NTPase activities are essential for viral replication (6), and the lack of HCV genotype-specific differences in helicase residues and activities (7), support a role for NS3 as an important pathogenic component of HCV. The identification of specific inhibitors of HCV NS3 helicase will add insights into the biology of HCV infection and replication, and serve as valuable tools for inhibiting HCV replication in human cells.

References:

1. Hoofnagle, J.H., Course and outcome of hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. s21-s29.
2. Frick, D.N., The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol, 2007. 9(1): p. 1-20.
3. Borowski, P., Schalinski, S., and Schmitz, H., Nucleotide triphosphatase/helicase of hepatitis C virus as a target for antiviral therapy. Antiviral Res, 2002. 55(3): p. 397-412.
4. Kim, J.L., Morgenstern, K.A., Griffith, J.P., Dwyer, M.D., Thomson, J.A., Murcko, M.A., Lin, C., and Caron, P.R., Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure, 1998. 6(1): p. 89-100.
5. Yang, J.P., Zhou, D., and Wong-Staal, F., Screening of small-molecule compounds as inhibitors of HCV entry. Methods Mol Biol, 2009. 510: p. 295-304.
6. Gu, B., Liu, C., Lin-Goerke, J., Maley, D.R., Gutshall, L.L., Feltenberger, C.A., and Del Vecchio, A.M., The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication. J Virol, 2000. 74(4): p. 1794-800.
7. Cho, H.S., Ha, N.C., Kang, L.W., Chung, K.M., Back, S.H., Jang, S.K., and Oh, B.H., Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem, 1998. 273(24): p. 15045-52.

Keywords:

late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, HCV, NS3, NS3 helicase, hepatitis, RNA virus, dose response, assay provider, inhibitor, inhibition, inhibit, Huh 7.5, HCV Rluc-replicon, total RNA, RT-PCR, qRT-PCR, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to assess the effect of compounds identified as possible probe candidates on RNA levels in Huh 7.5/HCV Rluc-replicon cells. Total RNA is extracted using TRizol total RNA extraction kit (Invitrogen) and RNA concentration is determined by reading absorbance at 260 nm. RT-PCR using TaqMan chemistry and the qScript(trade mark) One-step Fast qRT-PCR kit (Quanta Biosciences, Gaithersburg, MD) is used to quantify HCV RNA levels and the results are normalized to the ribosomal RNA.

Protocol Summary:

HCV RLuc replicon cells were seeded at a density of 2 x 10^5 cells per well in 6-well plates and incubated for 4-5 hours to allow the cells to attach to the plate. The compounds dissolved in dimethyl sulfoxide (DMSO) were added (DMSO solvent final concentration was 0.5%) and the cells were incubated for 72 hours at 37 C under 5% CO2 atmosphere. At the end of the treatment the cells were washed 2 times with ice cold phosphate buffered-saline and harvested by scraping and collected by low speed centrifugation at 1000 x g for 5 minutes at 4 C. Total RNA was then extracted using TRizol total RNA extraction kit (Invitrogen) following the manufacturer's instructions and suspended in 30 uL of nuclease-free water. RNA concentration was determined by reading the absorbance at 260 nm and RNA samples were stored in aliquots at -70 C until needed. Real time RT-PCR was performed with the TaqMan chemistry using 1 ug total RNA and the qScript(trade mark) One-step Fast qRT-PCR kit (Quanta Biosciences, Gaithersburg, MD) following the manufacturer's instructions. Reverse transcription was done at 50 C for 20 minutes followed by one cycle at 95 C for 5 minutes and 40 cycles at 95 C, 15 seconds and 60 C for 1 minute. HCV primers and probe target the HCV 5'UTR. The house-keeping gene rRNA was amplified in parallel for normalization. The expression levels of HCV RNA in each sample were determined by first calculating the delta_CT of each sample which was obtained by subtracting the threshold cycle (CT) of each sample including the DMSO-control sample from that of the corresponding housekeeping gene rRNA. The relative expression level was then determined by the 2^(-delta_delta_Ct) method where delta_delta_CT is the difference between the delta_CT of each sample and that of the DMSO-control sample. Dose response curves were fit to the data for IC50 determination using GraphPad Prism (v. 5).

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 50 uM were considered inactive. Compounds with an IC50 equal to or less than 50 uM were considered active.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.

List of Reagents:

HCV Rluc (gift from Seng-Lai Tan, supplied by Assay Provider)
Huh 7.5 cells (gift of Rice Lab at Rockefeller University, supplied by Assay Provider)
DMEM (Life Technologies, part 11995)
DMSO (Fisher Scientific, part BP231)
Geneticin (Life Technologies, part 10131)
TRizol Reagent (Life Technologies, part 15596-026)
aScript One-Step Fast qRT-PCR kit (Quanta Biosciences, part 95079-500)
6-well plates (Nunc, part 140675)
Comment
This assay was performed by the assay provider, and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased or synthesized by the University of Kansas Specialized Chemistry Center. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed in the Related Bioassays section of this AID.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: protein complex format
BAO: bioassay specification: assay biosafety level: bsl1
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: spectrophotometry: absorbance
BAO: meta target: biological process target: viral genome replication
BAO: meta target: molecular target: protein target: enzyme regulator
BAO: version: 1.4b1090
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HUH7
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identified if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2Average IC50*The average of the concentration at which 50 percent of the activity in the antagonist assay is observed (IC50); shown in micromolarFloatμM
3Standard ErrorStandard error of the average IC50.Float
4Average REL at 50 uM (50μM**)Value of average RNA relative expression level (REL) at 50 uM compound concentrationFloat%
5Average REL at 25 uM (25μM**)Value of average RNA relative expression level (REL) at 25 uM compound concentrationFloat%
6Average REL at 12.5 uM (12.5μM**)Value of average RNA relative expression level (REL) at 12.5 uM compound concentrationFloat%
7Average REL at 6.25 uM (6.25μM**)Value of average RNA relative expression level (REL) at 6.25 uM compound concentrationFloat%
8Average REL at 3.13 uM (3.13μM**)Value of average RNA relative expression level (REL) at 3.13 uM compound concentrationFloat%
9Average REL at 0 uM (0μM**)Value of average RNA relative expression level (REL) at 0 uM compound concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH085690-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: