Assay for stage-specific inhibitors of Vaccinia orthopox Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2142-01_Inhibitor_Dose_DryPowder_Activity
Assay Overview: Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia, the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox . Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, more ..
BioActive Compounds: 36
Keywords: Vaccina, orthopoxvirus
Assay Overview: Orthopoxviruses are a genus of viruses that include monkeypox, variola (the causative agent of smallpox) and vaccinia, the prototypical orthopoxvirus which was used in the world-wide vaccination program that eradicated smallpox . Smallpox was once the most deadly human pathogen, and is estimated to have killed more than 300 million people. Following the eradication of smallpox, routine vaccination was discontinued in the 1970s, and there has consequently been a precipitous decline in population immunity to smallpox and other orthopoxviruses. There are currently no FDA-licensed drugs to treat infected individuals, which is a significant concern given the threat of orthopoxvirus weaponization and the rise in reports of humans infected with monkeypox, which is endemic to Central and Western Africa . Potent and validated inhibitors of this assay will be of significant interest to public and will provide the development of initial compound leads into therapeutic agents, either as a single agent or as a combination therapy with existing anti-viral agents.
In collaboration with the Connor lab, a high throughput assay using vaccinia virus model system was developed. The assay used recombinant viruses (coupled reporter system) that robustly monitor viral gene expression and viral spread to identify novel anti-orthopoxviral compounds. The first infection evaluates the stage gene expression in Vaccinia using the fluorescent Venus protein while the late infection uses the fluorescent mCherry protein reporter.
Expected Outcome: Compounds that show a statistically significant inhibition of the mCherry late expression fluorescence signal, and that also show a significant window of lower inhibition of the Venus early expression fluorescence signal, will be considered active.
 Moss B (2007) Poxviridae: The Viruses and Their Replication. In: Knipe DMH, P.M., editor. Fields Virology. 5 ed: Lippincott Williams & Wilkins.
 Rimoin AW, Kisalu N, Kebela-Ilunga B, Mukaba T, Wright LL et al. (2007) Endemic human monkeypox, Democratic Republic of Congo, 2001-2004. Emerg Infect Dis 13(6): 934-937
A549 cells were plated at a concentration of approximately 6,000 cells per well in clear-bottom black-walled plates. Compounds were added to the assay plate followed by addition of virus (LREV) that expresses the Venus fluorescent protein early in viral infection. During the late viral infection, mCherry was also added to the plate at an MOI of 10 (~ 60,000 plaque forming units (PFU) of virus). Following virus addition, plates were incubated in a tissue-culture incubator for 12-18 hours. At increasing times post infection, fluorescence from both Venus and mCherrry was determined using a plate-reader and standard conditions for observing Venus fluorescence (EX515/EM530) and mCherry fluorescence (EX587/EM610). In each plate, positive controls (virus infection in the presence of 1% DMSO only) and an inhibitory drug control (AraC at 500 nM) was included and used to normalize the data.
1a, 4 uL A549 cell
1b 4 uL DMEM medium Control
1000 cell/well of A549 ad let cells attach for 3 hrs at 37C in CO2 incubator, DMEM medium in negative control columns
2a Control Inhibitor
2b 23 nL Offline Library#
23 nL Control Inhibitor, Intraplate 16-pt titration: AraC for mCherry detection & Actinomycin D for Venus detection
3 2 uL Viral Infection, 5 MOI of VAC-LREV virus in DMEM medium is dispensed by Bioraptor
4 Incubation for viral replication Overnight 37C in CO2 incubator
5 Envision Detection Over 18 hrs more Venus read: Exc 510nm/Em590nm & mCherry read: Exc 545nm/Em615nm
PRESENCE OF CONTROLS: Neutral control wells (NC; n=192) and positive control wells (PC; n=32) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated for each fluorescence channel using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome for each channel = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome for each channel= 2 (active) when:
AC <= AC_limit
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, selectivity was determined using the relative AC50s of the two channels. Compounds active in the mCherry channel and >5x less potent in the Venus channel are desired.
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when AC50 Venus > 5x AC50 mCherry, or when mCherry was active and Venus was inactive;
b. '1' (inactive) if both channels were inactive
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as value of the mCherry AC50
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE for active compounds was equal to the PUBCHEM_ACTIVITY_SCORE for the mCherry channel.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
* Activity Concentration.
Data Table (Concise)