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BioAssay: AID 492967

A screen for compounds that inhibit the CapD enzyme of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax. The enzyme CapD cleaves the poly-gamma-D-glutamate capsule and generates amide bonds with peptidoglycan crossbridges to deposit capsular material into the envelope of B. anthracis. B. anthracis capD variants do not deposit capsule into the envelope and display defects in anthrax pathogenesis. This screen identified the CapD inhibitor capsidin, more ..
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 Tested Compounds
 Tested Compounds
All(138861)
 
 
Active(320)
 
 
Inactive(138543)
 
 
 Tested Substances
 Tested Substances
All(139468)
 
 
Active(320)
 
 
Inactive(139148)
 
 
 Related BioAssays
 Related BioAssays
AID: 492967
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS645)
Depositor Category: Other
Deposit Date: 2010-12-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 320
Description:
Bacillus anthracis is the causative agent of anthrax. The enzyme CapD cleaves the poly-gamma-D-glutamate capsule and generates amide bonds with peptidoglycan crossbridges to deposit capsular material into the envelope of B. anthracis. B. anthracis capD variants do not deposit capsule into the envelope and display defects in anthrax pathogenesis. This screen identified the CapD inhibitor capsidin, 4-[(4-bromophenyl)thio]-3-(diacetylamino)benzoic acid), which covalently modifies the active site threonine of the transpeptidase. Capsidin treatment blocks capsular assembly by B. anthracis and enables phagocytic killing of non-encapsulated vegetative forms.
Protocol
Wells of a black polystyrene 384-well plate were loaded with 30 microL of a solution containing 20 pmol CapD in 25 mM HEPES-KOH, 0.1% Tween-20, pH 7.5.

Compounds were transferred to assay plates using a Seiko pin-transfer robot equipped with a stainless steel pin array. 100 nL of test compound was added to each well containing the assay mix and CapD. Pre-incubation with compound proceeded for 60 min at 22 degrees C. The reaction was then started by adding 400 pmol substrate in a 10 microL volume to each well.

After 90-120 minutes, assay plates were read using a PerkinElmer EnVision (excitation/emission 315/415 nm).
Comment
Raw data were filtered to exclude highly fluorescent compounds. The cutoff was derived from earlier validation experiments and defined as fluorescence intensity (FI) at saturation point plus 3 standard deviations; specifically, wells > 2,600,000 RFU for either replicate were excluded. Compounds in wells that exceeded the cutoff were considered auto-fluorescent (AF), and the FI values were excluded from plate means used in calculating Z-scores.

The experimental mean FI and standard deviation were calculated for each plate to derive a Z-score for every measurement using the equation Z = (X-Mean)/(SD). Wells were considered active if the Z-score for at least one replicate < -4.0. For each well and replicate, % activity was calculated by subtracting the well FI from the plate negative control average intensity, dividing by the difference between negative and positive control average plate intensity, and multiplying by 100. Activity scores were determined using average % activity of the two well replicates. Average % activity <= 0 was scored as 0 for activity; average % activity >= 100 was scored as 100 for activity. Average % activity values between 0 and 100 were used to generate activity scores for intermediate values. It should be noted that since Z-scores (not activity scores) were used to define positives, there is a general correlation but no strict relationship between activity scores and activity outcomes.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Intensity_ATotal fluorescence intensity for replicate AFloat
2Intensity_BTotal fluorescence intensity for replicate BFloat
3Z-score_AZ-score for replicate A, calculated using experimental well plate average FI and standard deviationFloat
4Z-score_BZ-score for replicate B, calculated using experimental well plate average FI and standard deviationFloat
5Auto-fluorescentIndicates whether a compound was considered auto-fluorescent (AF), based on the well total fluorescence intensityString

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