Late 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-NS4A
Name: Late 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-NS4A. ..more
BioActive Compounds: 9
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-NS4A_INH_QFRET_96_1XIC50
Name: Late 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-NS4A.
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.
8. Frick,D.N., Ginzburg,O. and Lam,A.M. (2010) A method to simultaneously monitor hepatitis C virus NS3 helicase and protease activities. Methods Mol Biol 587, 223-233.
late stage, late stage AID, powders, University of Kansas, University of Kansas Specialized Chemistry Center, KUSCC, KU, HCV, NS3, NS3 helicase, hepatitisRNA virus, dose response, 96, assay provider, inhibitor, inhibition, inhibit, fluorescence, HCV protease NS3-NS4A, FRET, QFRET, quench, quencher, QXL 520, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to verify enzyme activity of compounds and to examine specificity by determining whether powder samples of compounds identified as possible probe candidates inhibit the helicase encoded by the HCV protease NS3-NS4A. Upon cleavage of the substrate peptide by the NS3-NS4A protease, the 5-FAM fluorophore is separated from the QXL 520 quencher and fluorescence is recovered. As designed, compounds which inhibit the protease activity will result in no fluorescence increase.
All protease assays were carried out using the AnaSpec Enzolyte 520 Protease Assay Kit according to the manufacturer's protocol. Each assay contained either the test compound or DMSO as a negative control. Assays were carried out in a total volume of 20 uL in black 96-well half area plates with fluorescence measured using a FluoStar Omega (BMG Labtech). The protease enzyme used was 10 nM of "single chain" NS3-NS4A (scNS3-4A), where the portion of NS4A needed for protease activation is tethered to the N-terminus of NS3 (8). The concentration at which a compound causes a 50% reduction in reaction velocity (IC50) was calculated by fitting compound concentration to initial velocity using the equation:
vi = v0 / ( 1 + ( [I] / IC50 ) ^ h )
vi = initial reaction velocity
v0 = velocity observed in DMSO-only controls
[I] = compound concentration
h = Hillslope coefficient
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 100-48, and for inactive compounds 44-0.
List of Reagents:
NS3-NS4A (scNS3-4A) (Supplied by Assay Provider)
AnaSpec Enzolyte 520 Protease Assay Kit (AnaSpec, part 71145)
96-well plates (Corning Costar, black half volume, part 3694)
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: fluorescence: fluorescence intensity
BAO: meta target: biological process target: viral genome replication
BAO: meta target: molecular target: protein target: enzyme regulator
BAO: version: 1.4b1090
Assay Format: Biochemical
* Activity Concentration.
Data Table (Concise)