Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe more ..
BioActive Compounds: 41
Depositor Specified Assays
Description:
Emory Chemistry-Biology Discovery Center Assay Overview:
Grant number: none
Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for pre-existing or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals.
Grant number: none
Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for pre-existing or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals.
Protocol
Methods and Materials
Cell culture, transfection, and production of MV stocks. All cell lines were maintained at 37 deg C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. Vero-SLAM cells, derived from Vero (African green monkey kidney epithelial) cells (ATCC CCL-81) and stably expressing human SLAM/CD150w, Vero-dogSLAM cells stably expressing dog SLAM, and BSR T7/5 cells stably expressing T7 polymerase were incubated at every third passage in the additional presence of G-418 (Geneticin) at a concentration of 100 μg/ml. Lipofectamine 2000 Invitrogen) was used for transient transfection experiments. To prepare virus stocks, cells were infected at a
multiplicity of infection (MOI) of 0.001 plaque-forming units (pfu)/cell and incubated at 37 deg C.
Cells were scraped in OPTIMEM (Invitrogen), virus released by two freeze-thaw cycles, and titers determined by 50% tissue culture infective dose (TCID50) titration according to the Spearman-Karber method. MV-Edmonston (MV-Edm) stocks were grown and titered on Vero cells, while for MV field isolates Vero-SLAM cells and for canine distemper virus (CDV) Vero-dogSLAM cells were used. All MV field isolates were originally derived from PBMC samples and the viruses were isolated and minimally passaged on SLAM-positive B95-a cells or Vero-SLAM cells.
High throughput compound screening:
For screening, Vero cells were seeded in 96-well microtiter plates at a density of 7,500 cells per well in 100 ml growth medium. After a four-hour incubation period at 37 deg C and 5% CO2, compound was added in 1.0 μl/well doses (25 μM final concentration) with a Sciclone automated liquid handler system (Caliper, MA), followed by infection with rMV-eGFP at an MOI of 0.25 pfu/cell in 100 ml serum-free medium. Final solvent (DMSO) concentrations were 0.5% at which no adverse effect on cell viability or virus growth could be detected in control samples. All virus stocks 1 used for screening were subjected to dialysis against PBS to remove contaminating eGFP that has been synthesized during virus growth.
Following a 64-hour incubation period at 37 deg C, green fluorescence indicating expression of viral proteins was quantified using an Analyst HT microplate reader (Molecular Devices). To validate the assay, the MV fusion inhibitor AS-48 was added in an
otherwise identical setting and z' values were calculated according to the formula:
z' = 1- (3SD(C)+3SD(B))/(Mean(C)-Mean(B)), with C: control and B: background.
As first-pass test to exclude false-positive compounds, cytotoxicity was assessed microscopically for all wells that showed low fluorescence intensity and, for selected compounds, photo-documented at a10 magnification of 200x. The compound library used is a diversity set purchased by the Emory Chemical Biology Discovery Center from ChemDiv (San Diego, CA).
Data were normalized for deposition into PubChem using the number of standard deviations from the mean compound value per plate. The field SDs from Mean was calculated by the formula:
Y = (CompoundFluorescence - MeanCompoundFluorescence) / SDCompoundFluorescence
, where the mean and sd refer to the aggregate value for all compound wells on a plate.
PUBCHEM_ACTIVITY_SCORE was obtained by normalizing the number of SDs from the mean between the highest observed value for the assay and the lowest observed value, according to the formula:
SCORE = 100 * (Value - MaxVal) / (MinVal - MaxVal)
Compounds which showed a deviation of >3SDs below the mean value for the plate were marked as active, others were marked as inactive.
Cell culture, transfection, and production of MV stocks. All cell lines were maintained at 37 deg C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. Vero-SLAM cells, derived from Vero (African green monkey kidney epithelial) cells (ATCC CCL-81) and stably expressing human SLAM/CD150w, Vero-dogSLAM cells stably expressing dog SLAM, and BSR T7/5 cells stably expressing T7 polymerase were incubated at every third passage in the additional presence of G-418 (Geneticin) at a concentration of 100 μg/ml. Lipofectamine 2000 Invitrogen) was used for transient transfection experiments. To prepare virus stocks, cells were infected at a
multiplicity of infection (MOI) of 0.001 plaque-forming units (pfu)/cell and incubated at 37 deg C.
Cells were scraped in OPTIMEM (Invitrogen), virus released by two freeze-thaw cycles, and titers determined by 50% tissue culture infective dose (TCID50) titration according to the Spearman-Karber method. MV-Edmonston (MV-Edm) stocks were grown and titered on Vero cells, while for MV field isolates Vero-SLAM cells and for canine distemper virus (CDV) Vero-dogSLAM cells were used. All MV field isolates were originally derived from PBMC samples and the viruses were isolated and minimally passaged on SLAM-positive B95-a cells or Vero-SLAM cells.
High throughput compound screening:
For screening, Vero cells were seeded in 96-well microtiter plates at a density of 7,500 cells per well in 100 ml growth medium. After a four-hour incubation period at 37 deg C and 5% CO2, compound was added in 1.0 μl/well doses (25 μM final concentration) with a Sciclone automated liquid handler system (Caliper, MA), followed by infection with rMV-eGFP at an MOI of 0.25 pfu/cell in 100 ml serum-free medium. Final solvent (DMSO) concentrations were 0.5% at which no adverse effect on cell viability or virus growth could be detected in control samples. All virus stocks 1 used for screening were subjected to dialysis against PBS to remove contaminating eGFP that has been synthesized during virus growth.
Following a 64-hour incubation period at 37 deg C, green fluorescence indicating expression of viral proteins was quantified using an Analyst HT microplate reader (Molecular Devices). To validate the assay, the MV fusion inhibitor AS-48 was added in an
otherwise identical setting and z' values were calculated according to the formula:
z' = 1- (3SD(C)+3SD(B))/(Mean(C)-Mean(B)), with C: control and B: background.
As first-pass test to exclude false-positive compounds, cytotoxicity was assessed microscopically for all wells that showed low fluorescence intensity and, for selected compounds, photo-documented at a10 magnification of 200x. The compound library used is a diversity set purchased by the Emory Chemical Biology Discovery Center from ChemDiv (San Diego, CA).
Data were normalized for deposition into PubChem using the number of standard deviations from the mean compound value per plate. The field SDs from Mean was calculated by the formula:
Y = (CompoundFluorescence - MeanCompoundFluorescence) / SDCompoundFluorescence
, where the mean and sd refer to the aggregate value for all compound wells on a plate.
PUBCHEM_ACTIVITY_SCORE was obtained by normalizing the number of SDs from the mean between the highest observed value for the assay and the lowest observed value, according to the formula:
SCORE = 100 * (Value - MaxVal) / (MinVal - MaxVal)
Compounds which showed a deviation of >3SDs below the mean value for the plate were marked as active, others were marked as inactive.
Comment
Possible interference in this assay, but are not limited to, compounds with auto-fluorescence, compound quenching fluorescence, or precipitate.
Result Definitions
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Fluorescence | Fluorescence intensity, measured with an Analyst HT reader | | Integer | rsec | |
| 2 | SDs from Mean | The number of standard deviations from the mean compound value for the plate the sample was tested on | | Float | ||
| 3 | Plate Compound Fl Mean | Mean of fluorescence for the compound wells on the plate | | Float | rsec | |
| 4 | Plate Compound Fl SD | SD of the fluroescence for the compound wells on the plate | | Float | rsec |
Data Table (Concise)
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