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BioAssay: AID 912

qHTS Assay for Anthrax Lethal Toxin Internalization

Lethal factor (LF, 83 kDa), edema factor (98 kDa) and protective antigen (PA, 83 kDa) are lethal toxins produced by bacillus anthracis, a Gram-positive and spore-forming bacterium responsible for anthrax. While LF and EF contribute the cytotoxic activity of anthrax bacteria, PA is required for internalization of LF an EF. ..more
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 Tested Compounds
 Tested Compounds
All(68959)
 
 
Active(466)
 
 
Inactive(56456)
 
 
Inconclusive(12365)
 
 
 Tested Substances
 Tested Substances
All(70086)
 
 
Active(472)
 
 
Inactive(57122)
 
 
Inconclusive(12492)
 
 
 Related BioAssays
 Related BioAssays
AID: 912
Data Source: NCGC (ANTX642)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-19
Modify Date: 2010-07-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 466
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: 1 X01 MH082337-01
PI Name: Dr. Thomas Bugge

NCGC Assay Overview:

Lethal factor (LF, 83 kDa), edema factor (98 kDa) and protective antigen (PA, 83 kDa) are lethal toxins produced by bacillus anthracis, a Gram-positive and spore-forming bacterium responsible for anthrax. While LF and EF contribute the cytotoxic activity of anthrax bacteria, PA is required for internalization of LF an EF.

To enable translocation of anthrax toxins to the cytoplasm, PA has to be cleaved by cell surface furin or furin-like protease, forms a PA heptamer complex and binds to anthrax toxin receptor on cell-surface. Heptamerization of PA generates high-affinity binding sites for anthrax toxins, and upon binding to the toxin, is translocated to the cytoplasm. In addition, similar to many other bacterial toxins (e.g. diphtheria, botulinum, and tetanus toxins), a proton gradient is required for the anthrax toxin translocation. Low molecular weight molecules which interfere those internalization processes, may potentially lead to development of antidotes for the anthrax toxin.

By using LF-beta-lactamase fusion proteins, a quantitative high throughput assay was developed to screen small molecules potentially reducing or blocking internalization of the anthrax toxin. The fusion protein was generated by fusing the PA-binding region of LF N-terminal (1-254 a.a.) to the TEM-1 beta-lactamase (developed by Dr. Thomas Bugge's laboratory in NIH). In the presence of PA, LF-beta-lactamase fusion proteins will be internalized, and act on beta-lactamase substrate (CCF2/AM) trapped in cells after cleavage by cytoplasm esterases. An intact CCF2 fluoresces at 520 nm (green light) by FRET. CCF2 hydrolyzed by beta-lactamase releases acceptor fluorescence and fluoresces at 447 nm (blue light). EnVision plate reader (PerkinElmer, Boston, MA) was used to monitor fluoresce intensity with the following filter set, Lambda(EX)=405 nm, Lambda(EM)=460nm/530 nm. The data were plotted a ratio of the 460nm/530nm emissions.
Protocol
NCGC Assay Protocol Summary
ME-180 cells were maintained in F12 medium (Invitrogen, Carlsbad, CA) at 37C under a humidified atmosphere containing 5% CO2 and 95% air. The medium contained 10% FBS, 2 mM L-glutamine, 50 U/ml gentamicin (Invitrogen). The cells were stored at -135 C for the assay. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format plate (Kalypsys, treated black clear bottom) as follows:
1.2k/well frozen ME-180 was seed for overnight with 1% FBS at 37C under a humidified atmosphere containing 5% CO2 and 95% air.
2.Pinning 23 nL compounds. The final titration was 0.6 nM to 46 uM.
3.Incubation at 37C for 15 min.
4.Dispensing 1uL LF-beta-Lac (1 ug/ml final) with or without PA (2 ug/ml final).
5.Incubation at 37C for 2 hr.
6.Dispensing 1 uL of CCF2 in CCF2 detection buffer (CCF2, 0.5uM final)
7.Incubation at room temperature for 5 hr.
8.Reading plates by EnVision (PerkinElmer).
Keywords: Anthrax LF, LF internalization, beta-lactamase, CCF2, fluorescence, cytotoxicity, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
Comment
Compound Ranking:
1. Compounds are first classified as being probable inhibitors (compounds decreasing signal), activators (compounds increasing signal), fluorescent (artifactual results), cytotoxic (artifactual results), inactive, or inconclusive based on the ratio and component channel readouts.
2. Compounds are then classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Ratio-Curve Description".
3. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active inhibitor 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. Ratio-Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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
5Ratio-Curve_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 signficant, but below 80% of control.String
6Ratio-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Ratio-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Ratio-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Ratio-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Ratio-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Ratio-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Ratio-Excluded_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
13Ratio-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Ratio-Activity at 0.0005000000 uM (0.0005μM**)% Activity at given concentration.Float%
15Ratio-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
16Ratio-Activity at 0.00260 uM (0.0026μM**)% Activity at given concentration.Float%
17Ratio-Activity at 0.00570 uM (0.0057μM**)% Activity at given concentration.Float%
18Ratio-Activity at 0.013 uM (0.0128μM**)% Activity at given concentration.Float%
19Ratio-Activity at 0.029 uM (0.0286μM**)% Activity at given concentration.Float%
20Ratio-Activity at 0.064 uM (0.064μM**)% Activity at given concentration.Float%
21Ratio-Activity at 0.143 uM (0.1431μM**)% Activity at given concentration.Float%
22Ratio-Activity at 0.320 uM (0.3201μM**)% Activity at given concentration.Float%
23Ratio-Activity at 0.716 uM (0.7157μM**)% Activity at given concentration.Float%
24Ratio-Activity at 1.600 uM (1.6002μM**)% Activity at given concentration.Float%
25Ratio-Activity at 3.578 uM (3.578μM**)% Activity at given concentration.Float%
26Ratio-Activity at 8.001 uM (8.0005μM**)% Activity at given concentration.Float%
27Ratio-Activity at 17.89 uM (17.8891μM**)% Activity at given concentration.Float%
28Ratio-Activity at 40.00 uM (40μM**)% Activity at given concentration.Float%
29W460-Curve_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 signficant, but below 80% of control.String
30W460-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
31W460-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
32W460-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
33W460-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
34W460-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
35W460-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
36W460-Excluded_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
37W460-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
38W460-Activity at 0.0005000000 uM (0.0005μM**)% Activity at given concentration.Float%
39W460-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
40W460-Activity at 0.00260 uM (0.0026μM**)% Activity at given concentration.Float%
41W460-Activity at 0.00570 uM (0.0057μM**)% Activity at given concentration.Float%
42W460-Activity at 0.013 uM (0.0128μM**)% Activity at given concentration.Float%
43W460-Activity at 0.029 uM (0.0286μM**)% Activity at given concentration.Float%
44W460-Activity at 0.064 uM (0.064μM**)% Activity at given concentration.Float%
45W460-Activity at 0.143 uM (0.1431μM**)% Activity at given concentration.Float%
46W460-Activity at 0.320 uM (0.3201μM**)% Activity at given concentration.Float%
47W460-Activity at 0.716 uM (0.7157μM**)% Activity at given concentration.Float%
48W460-Activity at 1.600 uM (1.6002μM**)% Activity at given concentration.Float%
49W460-Activity at 3.578 uM (3.578μM**)% Activity at given concentration.Float%
50W460-Activity at 8.001 uM (8.0005μM**)% Activity at given concentration.Float%
51W460-Activity at 17.89 uM (17.8891μM**)% Activity at given concentration.Float%
52W460-Activity at 40.00 uM (40μM**)% Activity at given concentration.Float%
53W530-Curve_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 signficant, but below 80% of control.String
54W530-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
55W530-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
56W530-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
57W530-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
58W530-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
59W530-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
60W530-Excluded_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
61W530-Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
62W530-Activity at 0.0005000000 uM (0.0005μM**)% Activity at given concentration.Float%
63W530-Activity at 0.00110 uM (0.0011μM**)% Activity at given concentration.Float%
64W530-Activity at 0.00260 uM (0.0026μM**)% Activity at given concentration.Float%
65W530-Activity at 0.00570 uM (0.0057μM**)% Activity at given concentration.Float%
66W530-Activity at 0.013 uM (0.0128μM**)% Activity at given concentration.Float%
67W530-Activity at 0.029 uM (0.0286μM**)% Activity at given concentration.Float%
68W530-Activity at 0.064 uM (0.064μM**)% Activity at given concentration.Float%
69W530-Activity at 0.143 uM (0.1431μM**)% Activity at given concentration.Float%
70W530-Activity at 0.320 uM (0.3201μM**)% Activity at given concentration.Float%
71W530-Activity at 0.716 uM (0.7157μM**)% Activity at given concentration.Float%
72W530-Activity at 1.600 uM (1.6002μM**)% Activity at given concentration.Float%
73W530-Activity at 3.578 uM (3.578μM**)% Activity at given concentration.Float%
74W530-Activity at 8.001 uM (8.0005μM**)% Activity at given concentration.Float%
75W530-Activity at 17.89 uM (17.8891μM**)% Activity at given concentration.Float%
76W530-Activity at 40.00 uM (40μM**)% Activity at given concentration.Float%
77Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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