Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dong Hoon Chung, University of Louisville
Award: 1 R03 MH087448-01A1
Introduction
This assay is designed to evaluate the response of HEp-2 cells to selected compounds from anti-VEEV screening ( AID: 588727) in order to begin developing a cytotoxicity profile.

Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in DMEM with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37oC in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.05% trypsin-EDTA.

Assay Media - Preparation of Complete DMEM/F12: DMEM/F12 (Invitrogen, Cat. No.11320) was supplemented with 5mL of Pen/Strep (Invitrogen, Cat. No. 10378016), 5 mL of 200mM glutamine(Invitrogen, Cat No.25030-081), and 10 mL of HI-FBS was added per 500 mL of media.

Dose Response Compound Preparation: For dose response screening, compounds or carrier control (DMSO) were diluted to 3x in Complete DMEM/F12. Test compounds were serially diluted 1:2 resulting in an 8 point dose response dilution series. (final plate well concentration ranging from 50 uM to 0.39 uM and a final DMSO concentration of 0.25%). Thirty ul of each dilution was dispensed to assay plates (0.75% DMSO) in triplicate.
Control Drug: The positive control drug for this assay, MPA was solubilized in DMSO.

Preparation of HEp-2 cells: Cells were harvested and resuspended to 267,000 cells per ml in Complete DMEM/F12.

Assay Set up: Forty five ul of HEp-2 cell suspension (12,000 cells/well) were plated in clear bottom black well 96-well plates and the plates were incubated in an incubator at 37oC in a humidified 5% CO2 atmosphere. The next day, thirty uL of drugging media (0.75% DMSO) were added to the each wells and the plates were incubated at 37oC for an hour. Each well then received fifteen uL of Complete DMEM/F12 media. For the cell control wells, Complete DMEM/F12 media were added instead of virus solution. Drug plating was conducted using a EVO100, 96MAC (Tecan) and virus was added using MicroFlo (Biotek). The assay plates were incubated for five days at 37 degrees C, 5% CO2 and 90% relative humidity.

Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Synergy4 multi-label reader.

Data Analysis: 16 control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.

Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active" (toxic) and CC50 values were calculated. If the % viability at all doses was >70%, the compound was defined as "Inactive" (non-toxic).
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.

Phenotypic Screen: Yes

Multiplexing: No

BSL: BSL1

Screening Concentration Range Max: 50

Screening Concentration Range Min: 0.04

Assay Format: Cell-based

Assay Type: Viability/Toxicity

Assay Method: End-point

Assay Detection: Bio-luminescence

Used for Hit Validation?: No

Used during SAR?: Yes

Secondary Assay Sub-type: Counter-screen Assay

Grant Number: 1 R03 MH087448-01A1