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BioAssay: AID 685

Yeast Toxicity Assay.

eIF2B-related disorders are caused by genetically inherited mutations in the general translation initiation factor eIF2B [Pavitt GD, Ramaiah KV, Kimball SR, Hinnebusch AG, Genes Dev. 1998, 12, 514-26]. We are currently screening the MLSCN library to identify compounds that can restore eIF2B function using wild-type (WT) and mutant Saccharomyces cerevisiae as a cellular model for the disease. more ..
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 Tested Compounds
 Tested Compounds
All(30076)
 
 
Active(82)
 
 
Inactive(29893)
 
 
Inconclusive(101)
 
 
 Tested Substances
 Tested Substances
All(30077)
 
 
Active(82)
 
 
Inactive(29894)
 
 
Inconclusive(101)
 
 
 Related BioAssays
 Related BioAssays
AID: 685
Data Source: PCMD (YEAST TOXICITY)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-04-20
Modify Date: 2008-10-07

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 82
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Dr. Graham Pavitt, University of Manchester, U.K.
MLSCN Grant: X01-MH077608-01

eIF2B-related disorders are caused by genetically inherited mutations in the general translation initiation factor eIF2B [Pavitt GD, Ramaiah KV, Kimball SR, Hinnebusch AG, Genes Dev. 1998, 12, 514-26]. We are currently screening the MLSCN library to identify compounds that can restore eIF2B function using wild-type (WT) and mutant Saccharomyces cerevisiae as a cellular model for the disease. Results will be posted on PubChem upon completion of the screen. As a prelude to the eIF2B screen, we performed a high-throughput cell-toxicity assay to identify compounds from the library that may be toxic to yeast cells. The two yeast strains studied were wild-type S. cerevisiae and the mutant eIF2Bepsilon-R284H. The assay measures cell growth in the presence of test compounds; inhibition of cell growth is indicative of toxicity. Cell growth gives rise to an increase in turbidity, which is monitored by measurement of absorbance at 595 nm after growing the cells for 16 hrs at 30 C.
Protocol
Materials

Yeast cells (wild type and mutant) were supplied by Dr. Graham Pavitt and Dr Rogerio Alves de Almeida (University of Manchester) as frozen stocks, prepared as described below. YPD media (10 g/l yeast extract; 20 g/l bacto peptone) and Breathe-Easy sealing films were purchased from Sigma (Cat# Z380059). 384-well polypropylene plates were from Greiner (Cat# 781280) and 384-well clear plates were from Corning (Cat# 3702)

Mutant and wild type cell stocks were prepared and stored as follows:
1.Under sterile conditions yeast strain was taken from -80 freezer and streaked onto YPD plate. The plate was incubated at 30 C for 2-3 days
2.A single colony from the YPD plate was inoculated into 50 ml YPD media in a 250 ml Erlenmeyer flask and grown overnight at 30 C with vigorous shaking (200 rpm)
3.Cell concentration was determined after overnight growth by measurement of absorbance at 595 nm. The volume of cells required to give the necessary quantity in step 4 below was centrifuged at 3000 g for 5 minutes.
4.The cell pellet was resuspended in 1 ml of YPD+15% glycerol (A595 = 5.Cells were aliquoted (for future single use), frozen in dry ice and stored at -80 C.

Assay

Yeast cells were grown in 384-well polypropylene plates, sealed with Breathe-Easy membranes, at 30 C for 16 hrs. Absorbance was measured at 595 nm using a Perkin Elmer Envision spectrophotometer. HTS was performed using 25 uM compound as described below.

HTS protocol WT screen

1.Fill 384-well Greiner plates with 25 uL YPD media using Multidrop-384
2.Add 25 uL additional media to column 1 (blank)
3.Add 120 nL of compound (10 mM in DMSO) using Evolution pintool
4.Add 25 uL wild type yeast cells (stock diluted 1:500) to all columns except 1 and 23 using Multidrop-micro
5.Add 25 uL mutant yeast cells to column 23 using Multidrop-micro
6.Seal plates with Breathe-Easy sealing film
7.Incubate at 30 C for 16 hrs
8.Read absorbance (595 nm) on Envision reader


HTS protocol Mutant screen

1.Fill 384-well Greiner plates with 25 uL YPD media using Multidrop-384
2.Add 25 uL additional media to column 1 (blank)
3.Add 120 nL of compound (10 mM in DMSO) using Evolution pintool
4.Add 25 uL mutant yeast cells (stock diluted 1:500) to all columns except 1 and 23 using Multidrop-micro
5.Add 25 uL wild type yeast cells to column 23 using Multidrop-micro
6.Seal plates with Breathe-Easy sealing film
7.Incubate at 30 C for 16 hrs
8.Read absorbance (595 nm) on Envision reader

Data analysis

Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds (25 uM in 0.2% DMSO) in columns 3-22, controls (wild-type cells for WT screen and mutant yeast cells for mutant screen) in columns 2 and 24, reference yeast strain (wild- type cells for mutant screen and mutant cells for WT screen) in column 23, and blanks (YPD media) in column 1. Percent inhibition of growth was calculated for each compound from the signal in absorbance units (OD) and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))

The reference yeast strain (column 23) is not used in the calculation. It was used to monitor consistency of cell growth throughout the screen.
Comment
Activity scoring

Activity scoring was based on the percent inhibition of growth in both the wild-type and mutant yeast strains as follows:

(1) For percent inhibition between 0 and 100, score = lower value of percent inhibition between wild-type and mutant strains
(2) For negative percent inhibition, score = 0


Activity Outcome

Results from the wild type and mutant screens were compared to identify compounds that were toxic to both types of cells. Compounds that gave percent inhibition >30 in both the wild-type and mutant screens were judged to be confirmed actives as they were toxic to both the yeast strains used.

Compounds that gave a percent inhibition > 50 in one of the strains but showed percent inhibition < 30 in the other strain are reported as inconclusive. These compounds showed significant toxicity against one of the yeast strains, but this toxicity was not confirmed against the other strain.

All other compounds are reported as inactive. These compounds appeared to be minimally toxic to the yeast cells.

Analysis of screening results

Results of the HTS were as follows:

Hits (>30% inhibition in both wild type and mutant) = 82
Inactives (<30% inhibition in both wild type and mutant) = 29894
Inconclusives (>50% inhibition in one strain, but <30% inhibition in the other) = 101

Contributors

This assay was developed by Dr Rogerio Alves de Almeida and Dr Graham Pavitt, University of Manchester, UK and submitted to the PCMD (Scott Diamond, Director; University of Pennsylvania) by Dr. Graham Pavitt,. Further assay development and HTS were carried out by Nuzhat Motlekar, and data was submitted by Andrew Napper and Nuzhat Motlekar.

We would like to thank Dr. Mandar Ghatnekar and Rajaram Gurumurthi (Infosys Technologies Ltd.) for providing us with a customized tool for data analysis. Our thanks also go to Huiyan Jing for preparing the yeast cell stocks.

Correspondence
Please address correspondence to Andrew Napper (napper@seas.upenn.edu) or Graham Pavitt (graham.pavitt@manchester.ac.uk).
Categorized Comment - additional comments and annotations
From PubChem:
Assay Type: Toxicity
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1WT Percent inhibitionFloat%
2WT Percent inhibition absorbanceFloatAU
3WT Percent Inhibition Control absorbanceFloatAU
4WT Percent Inhibition Control SDFloatAU
5WT Percent Inhibition number of control wells Integer
6WT Percent Inhibition control percent CVFloat%
7WT Percent Inhibition Blank absorbanceFloatAU
8WT Percent Inhibition Blank SDFloatAU
9WT Percent Inhibition number of blank wellsInteger
10WT Percent Inhibition Blank percent CVFloat%
11WT Percent Inhibition signal-to-background ratioFloat
12WT Percent Inhibition Z-factorFloat
13MUTANT Percent inhibitionFloat%
14MUTANT Percent inhibition absorbanceFloatAU
15MUTANT Percent inhibition control absorbanceFloatAU
16MUTANT Percent inhibition control SDFloatAU
17MUTANT Percent inhibition number of control wellsInteger
18MUTANT Percent inhibition control percent CVFloat%
19MUTANT Percent inhibition blank absorbanceFloatAU
20MUTANT Percent inhibition blank SDFloatAU
21MUTANT Percent Inhibition number of blank wells Integer
22MUTANT Percent Inhibition blank percent CVFloat%
23MUTANT Percent Inhibition signa-to-background ratioFloat
24MUTANT Percent Inhibition Z-factorFloat

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