Late-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 cytotoxic
Name: Late-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 cytotoxic. ..more
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: AVA5CYTOX_INH_96_CC50
Name: Late-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 cytotoxic.
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.
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.
late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, NIAID, Antimicrobial Acquisition and Coordinating Facility, AACF, HCV, NS3, NS3 helicase, hepatitis, RNA virus, counterscreen, dose response, inhibitor, inhibition, inhibit, cytotoxicity, neutral red dye, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay, performed by NIAIDs Antimicrobial Acquisition and Coordinating Facility (AACF) (http://niaid-aacf.org/protocols/HCV.htm) is to determine cytotoxicity of samples of purchased compounds identified as possible probe candidates. In this assay, cytotoxicity is assessed by neutral red dye uptake. As designed, compounds that are cytotoxic will kill cells and decrease the amount of red dye taken up.
Cytotoxicity is assessed in an assay using the stably-expressing HCV replicon cell line, AVA5 (sub-genomic [CON1], genotype 1b) maintained as sub-confluent cultures on 96-well plates. Cytotoxicity is assessed by neutral red dye uptake.
CC50 values are calculated by linear regression analysis (MS EXCEL(R), QuattroPro(R)) using data combined from all treated cultures. CC50 is the drug concentration at which a 2-fold lower level of neutral red dye uptake (relative to the average levels in untreated cultures) is observed.
PubChem Activity Outcome and Score:
Compounds with an CC50 greater than 10 uM were considered inactive. Compounds with an CC50 equal to or less than 10 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 inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Reagents were provided by the AACF program.
This assay was performed by the NIAIDs Antimicrobial Acquisition and Coordinating Facility (AACF), and submitted to PubChem by the Scripps Research Institute Molecular Screening Center (SRIMSC). Compounds tested in this assay were purchased 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.
* Activity Concentration.
Data Table (Concise)