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BioAssay: AID 651654

HTS for the detection of C. neoformans cell lysis via adenylate kinase (AK) release Measured in Microorganism System Using Plate Reader - 2162-01_Inhibitor_SinglePoint_HTS_Activity

Assay Overview: This is a novel HTS assay that specifically detects molecules that directly kill Cryptococcus yeast cells through the detection of AK released into the growth medium by cells that have undergone lysis. The assay is a simple "add and read" assay that uses a commercially available adenylate kinase detection cocktail (ToxiLight, Lonza, Cambridge, MA). The compounds are charged into more ..
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 Tested Compounds
 Tested Compounds
All(357995)
 
 
Active(735)
 
 
Inactive(357282)
 
 
 Tested Substances
 Tested Substances
All(361009)
 
 
Active(735)
 
 
Inactive(360274)
 
 
 Related BioAssays
 Related BioAssays
AID: 651654
Data Source: Broad Institute (2162-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-19

Data Table ( Complete ):           Active    All
BioActive Compounds: 735
Depositor Specified Assays
AIDNameTypeComment
651665Broad Institute Discovery and evaluation of fungicidal anti-cryptococcal molecules Inhibitor Probe Projectsummary
Description:
Keywords: Cryptococcus neoformans, adenylate kinase, cell wall integrity, antifungal


Assay Overview: This is a novel HTS assay that specifically detects molecules that directly kill Cryptococcus yeast cells through the detection of AK released into the growth medium by cells that have undergone lysis. The assay is a simple "add and read" assay that uses a commercially available adenylate kinase detection cocktail (ToxiLight, Lonza, Cambridge, MA). The compounds are charged into yeast medium (12.5 uL total volume, Yeast Peptone Dextrose) Initial work with S. cerevisiae revealed that the majority of molecules identified by the AK assay target either the plasma membrane or the cell wall. In order to distinguish between these two broad modes of action, a simple counter-screen allows us to identify cell wall targeted molecules.


Expected Outcome: increase of signal.
Protocol
1) Start overnight culture in YPD, 30 degrees C, 150 RPM. Dilute stock: 5 uL in 100 uL media, then transfer 5 uL to 20 ml for o/n culture.
2) Check OD of shake culture. This MUST be log phase, <1.
3) Spin culture to remove old media which will contain AK. Pour off old media and resuspend in fresh media. Repeat.
4) Do cell count on Cellometer. Adjust culture to 5x10e6 cells/ml.
5) Add 30 uL/well culture to assay plates (white 1536 Aurora HiBase) to which compound has been added (ARPs).
6) Add 0.5 uL/well positive control to poscon wells: 50 uM Clomiphene.
7) Incubate at 37 degrees C stat for 4 hours.
8) Take out ToxiLight to warm to room temp. ToxiLight should not sit at r/t any longer than necessary.
9) Fifteen minutes before dispensing, mix ToxiLight reagents and allow to equilibrate.
10) After assay plates have incubated for 4 hours, add 30 uL/well ToxiLight.
11) Shake (1500 rpm) for at least 5 min (30 min is ideal). Keep at room temp until read. Read at 1 hr after reagent addition.
12) Read is done on Perkin/Elmer Envision using UltraSensitive Luminescence setting.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 80.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 50.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_14.98uM_(%) (14.98μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_14.98uM_(%) (14.98μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: R01 AI091422

Data Table (Concise)
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