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
BioActive Compound: 1
Depositor Specified Assays
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
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, 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.
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.
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)
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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: protein complex format
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: meta target: molecular target: protein target: enzyme regulator
BAO: meta target: biological process target: viral genome replication
BAO: detection technology: spectrophotometry: absorbance
* Activity Concentration. ** Test Concentration.
Data Table (Concise)