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BioAssay: AID 2314

Cycloheximide Counterscreen for Small Molecule Inhibitors of Shiga Toxin

Shiga toxin (Stx) is released by certain strains of E. coli and is associated with food-borne gastroenteritis. In some patients, especially children, the toxin enters the bloodstream and causes hemolytic uremic syndrome, a condition that results in kidney, heart, and occasionally brain injury. The pathogenic effects of Stx arise by the toxin entering cells and inhibiting protein synthesis. To more ..
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 Tested Compounds
 Tested Compounds
All(295626)
 
 
Active(37013)
 
 
Inactive(258658)
 
 
 Tested Substances
 Tested Substances
All(296456)
 
 
Active(37055)
 
 
Inactive(259401)
 
 
AID: 2314
Data Source: NCGC (CHX001)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-01
Modify Date: 2010-02-04

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 37013
Related Experiments
AIDNameTypeComment
2315A qHTS for Small Molecule Inhibitors of Shiga ToxinScreeningdepositor-specified cross reference
2318Cycloheximide Counterscreen for Small Molecule Inhibitors of Shiga Toxin (Confirmatory)Confirmatorydepositor-specified cross reference
2320A qHTS for Small Molecule Inhibitors of Shiga Toxin (Confirmatory)Confirmatorydepositor-specified cross reference
2342A qHTS for Small Molecule Inhibitors of Shiga Toxin (Summary)Summarydepositor-specified cross reference
493122Concentration response cycloheximide confirmation counterscreen for small molecule inhibitors of Shiga toxinConfirmatorysame project related to Summary assay
493123Concentration response confirmation assay for small molecule inhibitors of Shiga toxinConfirmatorysame project related to Summary assay
Description:
Shiga toxin (Stx) is released by certain strains of E. coli and is associated with food-borne gastroenteritis. In some patients, especially children, the toxin enters the bloodstream and causes hemolytic uremic syndrome, a condition that results in kidney, heart, and occasionally brain injury. The pathogenic effects of Stx arise by the toxin entering cells and inhibiting protein synthesis. To identify inhibitors of Stx activity and transport, a cell-based assay was used that employed luciferase activity as a measure of protein synthesis in Stx-treated cells. Vero cells were transduced with an adenovirus expressing a form of luciferase that is degraded rapidly by the proteasome (pAd-luc). Exposure to Stx, which inhibits protein synthesis, results in markedly diminished luciferase translation (Zhao and Haslam, 2005; Saenz et al., 2007). A counterscreen assay was used to identify nonspecific compounds that inhibit protein synthesis independent of Stx action. Cycloheximide (Chx) readily diffuses into cells and immediately blocks protein synthesis in the absence of Stx. Stx actives that are active in the Chx assay are thus nonspecific compounds that stabilize luciferase activity.

Zhao L, Haslam DB. A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis. J Med Microbiol. 2005, 54:1023-30.

Saenz JB, Doggett TA, Haslam DB. Identification and characterization of small molecules that inhibit intracellular toxin transport. Infect Immun. 2007, 75:4552-61.
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Protocol
Six million Vero cells were seeded in a T225 flask and grown 24 hrs at 37 C and 5% CO2. The medium was replaced and the cells infected with 3 x109 plaque forming units of pAd-luc for 24 hrs at 37 C and 5% CO2. Cells were then suspended and dispensed at 750 cells/5 uL/well into white solid 1536-well plates (Grenier) using a solenoid-based dispenser. Following a 24 hr incubation at 37 C and 5% CO2, 23 nL compound or DMSO vehicle was added to each well by a pin tool, the plates were incubated 30 min at 37 C and 5% CO2, and 1 uL/well cyclohemixide (1 ug/mL final concentration) was added. The plates were centrifuged 30 s at 1000 RPM and incubated 6 hr at 37 C and 5% CO2. After addition of 3 uL Photinus luciferase detection reagent, the plates were incubated 10 min at ambient temperature and then read by an ViewLux (Perkin Elmer) to detect luminescence
Comment
1. A compound was active if its signal was greater than or equal to 3 standard deviations above the mean DMSO signal on its plate
2. Otherwise a compound is marked inactive
Active compounds receive a score of 100 and inactive compounds receive a score of 0
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: Vero
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
2PotencyConcentration 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
5Curve_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
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
15Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
16Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

** Test Concentration.
Additional Information
Grant Number: R03-MH081267-01

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