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BioAssay: AID 1830

Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).

Name: Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3). ..more
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AID: 1830
Data Source: The Scripps Research Institute Molecular Screening Center (NS3HELICASE_INH_ LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-24
Modify Date: 2012-07-25
Target
Related Experiments
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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)
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 (FID))
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 assay (Revoked and replaced by AID 588360)
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, set 2)
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 depositor-specified cross reference: Late stage dose response counterscreen (Helicase encoded by HCV strains inhibitors, set 3)
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 depositor-specified cross reference: Late stage counterscreen (cytotoxicity)
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 depositor-specified cross reference: Late stage assay (HCV replication inhibitors)
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 depositor-specified cross reference: Late stage assay (HCV protease NS3-NS4A inhibitors)
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 depositor-specified cross reference: Late stage assay (E. coli single stranded DNA binding protein displacement)
623968Late 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 levelsConfirmatory depositor-specified cross reference: Late stage assay (RNA levels effect)
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 depositor-specified cross reference: Late stage counterscreen (DNA binding with SYBR)
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 depositor-specified cross reference: Late stage counterscreen (DNA binding with EtBr)
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 depositor-specified cross reference: Late stage assay (ATPase activity inhibitors)
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: NS3HELICASE_INH_ LEADS_SUMMARY

Name: Summary of probe development efforts to identify inhibitors of the Hepatitis C Virus non-structural protein 3 helicase (NS3).

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.

Summary of Probe Development Effort:

Following primary HTS in singlicate to identify HCV NS3 helicase inhibitors (AID1800), compounds were identified as possible candidates for probe development. A probe development effort is currently underway at the SRIMSC.

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:

Summary AID, HCV, NS3 helicase, hepatitis, RNA virus, primary screen, HTS, high throughput screen, 1536, inhibitor, FLINT, fluorescence, fluorescence intensity, FLINT, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Please see AID 1800 for protocols performed in this probe development effort.
Comment
This probe development project is still underway at the SRIMSC.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML283
PubChem Substance ID (SID) for probe 1: 50930730
PubChem Compound ID (CID) for probe 1: 2739932
Probe type for probe 1: Inhibitor
Target for probe 1: HCV NS3 helicase (gi: 125541954)
Disease relevance for probe 1: Hepatitis C
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK143540/ (ID: 3035886)
Grant number for probe 1: MH085690-01
NCBI Book chapter title for probe 1: Hepatitis C Virus NS3 Helicase Inhibitor Discovery
Additional Information
Grant Number: 1 R03 MH085690-01

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