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BioAssay: AID 720580

qHTS for Stage-Specific Inhibitors of Vaccinia Orthopoxvirus: Venus Reporter Primary qHTS

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 [1]. 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 more ..
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 Tested Compounds
 Tested Compounds
All(326356)
 
 
Active(1520)
 
 
Inactive(305117)
 
 
Inconclusive(20107)
 
 
 Tested Substances
 Tested Substances
All(330461)
 
 
Active(1530)
 
 
Inactive(308714)
 
 
Inconclusive(20217)
 
 
 Related BioAssays
 Related BioAssays
AID: 720580
Data Source: NCGC (Vaccinia-p2Venus)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-08-19

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1520
Depositor Specified Assays
AIDNameTypeComment
720581qHTS for Stage-Specific Inhibitors of Vaccinia Orthopoxvirus: Summarysummary
Description:
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 [1]. 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 [2]. 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 quantitative 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. The data herein is the result of the primary screen using the Venus protein reporter. Decrease in signal in both reporters, indicative of orthopoxvirus inhibition is the desired outcome for this assay.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH094169
Assay Submitter (PI): John Connor, Boston University

[1] Moss B (2007) Poxviridae: The Viruses and Their Replication. In: Knipe DMH, P.M., editor. Fields Virology. 5 ed: Lippincott Williams & Wilkins.

[2] 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
Protocol
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.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Activity_ScoreActivity score.Integer
6Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.00111 uM (0.00111378μM**)% Activity at given concentration.Float%
16Activity at 0.00284 uM (0.00283798μM**)% Activity at given concentration.Float%
17Activity at 0.00446 uM (0.00445512μM**)% Activity at given concentration.Float%
18Activity at 0.00891 uM (0.00891024μM**)% Activity at given concentration.Float%
19Activity at 0.015 uM (0.0151706μM**)% Activity at given concentration.Float%
20Activity at 0.029 uM (0.0291896μM**)% Activity at given concentration.Float%
21Activity at 0.036 uM (0.035641μM**)% Activity at given concentration.Float%
22Activity at 0.073 uM (0.0728102μM**)% Activity at given concentration.Float%
23Activity at 0.113 uM (0.113104μM**)% Activity at given concentration.Float%
24Activity at 0.149 uM (0.149181μM**)% Activity at given concentration.Float%
25Activity at 0.293 uM (0.292539μM**)% Activity at given concentration.Float%
26Activity at 0.487 uM (0.486758μM**)% Activity at given concentration.Float%
27Activity at 0.726 uM (0.726486μM**)% Activity at given concentration.Float%
28Activity at 1.458 uM (1.45802μM**)% Activity at given concentration.Float%
29Activity at 2.011 uM (2.01123μM**)% Activity at given concentration.Float%
30Activity at 3.331 uM (3.33072μM**)% Activity at given concentration.Float%
31Activity at 5.199 uM (5.19888μM**)% Activity at given concentration.Float%
32Activity at 7.343 uM (7.34305μM**)% Activity at given concentration.Float%
33Activity at 13.78 uM (13.7772μM**)% Activity at given concentration.Float%
34Activity at 20.90 uM (20.8952μM**)% Activity at given concentration.Float%
35Activity at 36.52 uM (36.525μM**)% Activity at given concentration.Float%
36Activity at 60.71 uM (60.7051μM**)% Activity at given concentration.Float%
37Activity at 87.88 uM (87.8788μM**)% Activity at given concentration.Float%
38Activity at 127.3 uM (127.334μM**)% Activity at given concentration.Float%
39Activity at 194.0 uM (194μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH094169

Data Table (Concise)
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