A CPE Based HTS Assay for Antiviral Drug Screening Against Dengue Virus
Assay Rationale and Summary: Recently, dengue disease is the most important mosquito-borne viral disease affecting humans and is endemic in most tropical countries of the South Pacific, Asia, the Caribbean, the Americas and Africa. The recent trend of increased epidemic dengue activity and increased incidence of DHF around the world is unprecedented. In the Americas as of December 14, there were more ..
BioActive Compounds: 318
Depositor Specified Assays
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Centers Network (MLPCN)
Assay Provider: Dr. Qianjun Li, University of Alabama at Birmingham
Assay Rationale and Summary: Recently, dengue disease is the most important mosquito-borne viral disease affecting humans and is endemic in most tropical countries of the South Pacific, Asia, the Caribbean, the Americas and Africa. The recent trend of increased epidemic dengue activity and increased incidence of DHF around the world is unprecedented. In the Americas as of December 14, there were 893,453 reported cases, including 23,980 DHF cases and 370 deaths in 2009 (32, 33). There is a small risk for dengue outbreaks in the continental United States, and most dengue cases in the US Continental are travel-associated, however, DF cases were reported in Key West in July, 2009, which represents the 1st documented presence of dengue in Florida in more than 40 years (8). Dengue also occurs as endemic transmission among residents in the US territories (1). In 2009, a total of 5,788 suspected dengue cases, including 54 DHF cases and 2 deaths, were reported in Puerto Rico (33). With the rapid expansion of dengue disease in most tropical and subtropical areas of the world and the threats in Continental US, it is urgent to develop effective prevention and control measures, including antivirals and vaccines, for dengue disease. In the past few decades, dengue vaccines, including live-attenuated live tetravalent vaccines, have been developed, and several vaccine candidates have completed phase two trials, however, no FDA licensed dengue vaccine is currently available. Many factors contribute to the elusiveness of dengue vaccine, including the complexity of the dengue disease, such as "the antibody-dependent enhancement", the possible recombination between a vaccine strain and wild type virus, and a lack of the proper animal disease models for pathogenesis studies (13, 25). Recently, more attention has been paid to antiviral drug discovery against dengue disease. Several assays and strategies have been developed to identify potential antivirals, including virtual screening, screening of small-molecule compounds libraries using replicon-based assay, and antisense RNA strategies (22, 23, 36). A number of compounds have shown their efficacy in inhibiting DENV replication in vitro, including ribavirin, mycophenolic acid (MPA), 7-deaza-2'-Cmethyl-adenosine, and 6-O-butanoyl castanospermine (1, 5, 7, 24, 29-31, 34). Nevertheless, there are still no antiviral drugs being tested against dengue disease in any clinical trials.
With the rapid expansion of dengue disease in most tropical and subtropical areas of the world, it is urgent to develop antiviral drugs against dengue infection. Unfortunately, there are no efficient vaccines and no effective antiviral drugs available against DENV. Currently, vector control remains as the major prevention measure. Antiviral drug discovery requires the development of reliable, robust and HTS amenable biological assay(s), which is not available for DENV currently.
Cell Culture: BHK-21(C-13) cell line (ATCC CL-10) were propagated in cDMEM and incubated at 37.0 degrees C with 5% CO2 and high humidity. To subculturing the BHK-21 cells, remove medium, and rinse with 0.25% trypsin, 0.03% EDTA solution. Remove the solution and add an additional 1 to 2 ml of trypsin-EDTA solution. Allow the flask to sit at room temperature (or at 37C) until the cells detach. Add fresh culture medium, aspirate and dispense 1:5 into new culture flasks 2 to 3 times per week.
Assay Media - Preparation of Complete DMEM: Per 1 Liter of DMEM purchased from Invitrogen (Cat 11995), 40 mL of heat inactivated FBS (Invitrogen Cat 10082), 10 mL of HEPES (Invitrogen Cat 15630) and 10 mL Pen/Strep/Glutamine (Gibco Cat 15140) was added to generate complete media.
Infectious material - Dengue virus 2 (New Guinea C) (DENG2) purchased from ATCC (VR-1584). Viral stocks were maintained at -80C. The DENG2 titer as determined by TCID50 was 10;5.58 TCDI50/ml using BHK-21 cells.
Single Dose Compound Preparation: The MLS 10K was screened in a single dose format at 20uM final concentration and .2% DMSO. 3uL of compounds were dispensed into black clear-bottom TCT (Corning 3712) plates and the volume was adjusted with 7uL of cDMEM for a total of 10uL of compound. ##
Control Drug: Ribavirin was used at a positive control for the assay at a final concentration of 15uM and .2% DMSO.
Primary Assay Set up: BHK21 (C-13) cells were harvested and the concentration adjusted to 50,000 cells/ml in cDMEM. 20uL of BHK-21 (C-13) cells were dispensed using a WellMate (Matrix, Hudson, NH) to columns one and two of the black clear-bottom TCT (Corning 3712) plates for a negative cell control. The DENG2 was added to the BHK-21 (C-13) cells at a ratio of 1:50 for a final 1:75 dilution/well. Twenty microliters were dispensed to columns 3-24 of the plate. The plates were placed incubated at 37.0 degrees C with 5% CO2 and high humidity for four days.
Endpoint Read: Following the six day incubation period, the assay plates were equilibrated to room temperature for 30 min and an equal volume (30 muL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer EnvisionTM multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s.
Data Analysis: Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). thirty two control wells containing cells only and twenty four wells containing cells and virus were included on each assay plate and used to calculate Z' value for each plate and to normalize the data on a per plate basis. The overall Z score for the campaign were >/= 0.7. Results are reported as percent (%) CPE inhibition and were calculated using the following formula: % CPE inhibition = 100*(Test Cmpd - Med Virus)/(Med Cells - Med Virus). Eight ribavirin positive control wells were included on each plate for quality control purposes, but were not used in Z' calculations.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Active compounds showed Inhibition of CPE greater than 13.25% (the mean + 3 standard deviations of all compound data).
Compounds in the primary (single dose) screen were scored on a scale of 0-40, based on the maximum % CPE inhibition observed for the compound. On this scale, 0 corresponds to no inhibitive activity and a score of 40 corresponds to 100% CPE inhibition.
Phenotypic Screen: Yes
Screening Concentration: 20
Assay Format: Cell-based
Assay Type: Viability/Toxicity
Assay Method: End-point
Target Type: Viral Protein
Assay Detection: Bio-luminescence
Used for Hit Validation?: Yes
Used during SAR?: No
Data Table (Concise)