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BioAssay: AID 792

eIF2B IC50 against mutant and WT yeast

eIF2B is a translation initiation factor that functions in the first step of protein synthesis. It is a guanine-nucleotide exchange factor (GEF), converting eIF2 (inactive GDP-bound form) to eIF2GTP (active). Mutations of eIF2B manifest as a dysfunction in brain myelin leading to its progressive loss. This causes fatal conditions known as Childhood Ataxia with CNS Hypomyelination syndrome (CACH) more ..
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 Tested Compounds
 Tested Compounds
All(448)
 
 
Inactive(427)
 
 
Inconclusive(21)
 
 
 Tested Substances
 Tested Substances
All(448)
 
 
Inactive(427)
 
 
Inconclusive(21)
 
 
 Related BioAssays
 Related BioAssays
AID: 792
Data Source: PCMD (eIF2B IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-08-22
Modify Date: 2008-10-07

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
688Yeast eIF2B assayScreeningdepositor-specified cross reference: eIF2B single point screen for identifying hits
Description:
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD), University of Pennsylvania
Assay Provider: Dr. Graham Pavitt, University of Manchester, U.K.
MLSCN Grant: X01-MH077608-01


eIF2B is a translation initiation factor that functions in the first step of protein synthesis. It is a guanine-nucleotide exchange factor (GEF), converting eIF2 (inactive GDP-bound form) to eIF2GTP (active). Mutations of eIF2B manifest as a dysfunction in brain myelin leading to its progressive loss. This causes fatal conditions known as Childhood Ataxia with CNS Hypomyelination syndrome (CACH) and leukoencephalopathy with vanishing white matter (VWM). As eIF2B is a universally conserved protein among eukaryotic organisms, a cellular assay system has been developed using yeast, Saccharomyces cerevisiae (wild type and eIF2B-epsilon-R284H mutant). Yeast eIF2B-epsilon-R284H is equivalent to the human eIF2B5-R299H mutation. The HTS is aimed at identifying compounds that can restore eIF2B function.

The screen is a reporter-gene assay. GCN4 activity is inversely proportional to eIF2B function in cells. Thus cells with eIF2B mutations exhibit aberrantly high GCN4 expression. The mutant cells contain a chromosomally-integrated gene-fusion to beta-galactosidase. Beta-Galactosidase monitoring thus indirectly measures GCN4 levels and, in turn, eIF2B activity. To perform the assay, we have adapted a coupled beta-galactosidase/ firefly luciferase system (Promega) for use with yeast cells and in HTS format. The growth of mutant and wildtype yeast in the presence of each compound was also monitored and used as a measure of compound toxicity.

The HTS has been reported earlier (AID 688). Here we report the follow-up dose-response testing on the 448 compounds identified as hits in the HTS.
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; 20 g/l glucose), Breathe-Easy sealing films (Cat# Z380059) and buffer salts were purchased from Sigma. 384-well polypropylene plates were from Greiner (Cat# 781280) and 384-well clear plates (Cat# 3702) and white plates (Cat# 3702) were from Corning. Protease inhibitor tablets were from Roche (Cat# 1873580). Beta-Glo assay system was from Promega (Cat# E4780).
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 to A595 = 5. Cells were aliquoted (for future single use), frozen in dry ice and stored at -80 C.
Assay
Mutant yeast cells were grown in 384-well polypropylene plates sealed with Breathe-Easy membranes at 30 C for 16 hrs. After 16 hrs, the cells were mixed using Perkin Elmer Evolution by repeated aspirate-dispense cycles using 30 uL disposable tips. A portion of the cell suspension was lysed with Z-buffer (82 mM disodium hydrogen phosphate, 9 mM sodium dihydrogen phosphate, 0.1% SDS) in a white 384-well plate. Beta-Glo reagent was added to the lysed cells, and luminescence was measured after 90 min. at room temperature. The remaining cells were transferred to a clear 384-well plate and absorbance was measured at 595 nm using a Perkin Elmer Envision spectrophotometer. HTS was performed using 25 uM compound as described below. The screen was repeated using the wild-type yeast cells as a filter to identify compounds having a non-specific effect on the luminescence of both mutant and wild-type yeast.
Dose-response plate preparation
Serial dilute single compounds at 100x concentration in DMSO (16 two-fold dilutions from 10 mM to 305 nM)
IC50 protocol: GROWTH
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 0.5 uL of compound (10 mM in DMSO) using Evolution tips
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
IC50 protocol: LUMINESCENCE AND ABSORBANCE
1. Mix cells in 384-well Greiner plates using 30 uL disposable tips on Evolution
2. Add 20 uL Z-buffer to 384-well white plate using Multidrop-micro
3. Transfer 5 uL cells from Greiner plate into white plate using Evolution
4. Incubate white plate for 20 min (for cell lysis) at room temperature
5. Add 25 uL Beta-Glo reagent to lysed cells using Multidrop-384
6. Incubate at room temperature for 90 minutes
7. Read Luminescence using Envision reader
8. Transfer 30 uL cells from Greiner plate to Clear 384-well plate using Evolution
9. Measure Absorbance at 595 nm using Envision reader
Data analysis
Data were analyzed in IDBS ActivityBase. Each HTS plate contained compounds (25 uM in 0.25% 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 as well as percent inhibition of luminescence was calculated for each compound from the signal in absorbance units (OD)/luminescence units (LU) 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
Luminescence results from the mutant and wild-type screens were compared to identify compounds that selectively inhibit luminescence of mutant cells. The mutant growth assay was used to rule out toxic compounds.
The activity score reported here is based on follow-up IC50 testing on compounds that showed >50% inhibition in the mutant luminescence, < 30% inhibition in wild-type luminescence and < 30% inhibition in mutant growth.
IC50 score = (IC50 score #1)- (IC50 score #2)- (IC50 score #3)
where:
IC50 score #1 is calculated from the mean IC50 of the mutant luminescence assay
IC50 score #2 is calculated from the mean IC50 of the mutant growth assay
IC50 score #3 is calculated from the mean IC50 of the wild-type luminescence assay
All negative scores are reported as 0
IC50 scores were calculated as follows:
(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >100 uM (zero in IC50 column), score was calculated from percent activity at maximum concentration tested in assay (100 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]
A positive score would indicate an ACTIVE compound.
Activity Outcome
Compounds that were judged to be hits in the primary HTS were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >100 uM. The combined score takes into account the inhibition of mutant luminescence, inhibition of mutant growth (toxic compound) and inhibition of WT luminescence (non-specific inhibition).
Activity outcome is reported as follows:
(1) IC50 <100 uM in mutant luminescence and mutant growth, and IC50>100 uM in WT luminescence = ACTIVE
(2) IC50 <100 uM in mutant luminescence and mutant growth = TOXIC, hence not tested in WT luminescence
(3) IC50 <100 uM in mutant luminescence and WT luminescence = INACTIVE (non-specific inhibition)
Analysis of screening results
Results of retesting the hits identified in the primary HTS were as follows:
Hits = 448
Hits active in IC50 = 0(0% retest rate)
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 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 direct correspondence to Andrew Napper (napper@seas.upenn.edu).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierString
2mutant luminescence Mean IC50*FloatμM
3mutant luminescence standard deviationFloatμM
4number of IC50 determinationsInteger
5QualifierString
6mutant luminescence IC50 # 1FloatμM
7mutant luminescence IC50 # 1 Hill slopeFloat
8mutant luminescence IC50 # 1 R-squaredFloat
9mutant luminescence IC50 # 1 min concentrationFloatμM
10mutant luminescence IC50 # 1 percent activity at min concentrationFloat%
11mutant luminescence IC50 # 1 max concentrationFloatμM
12mutant luminescence IC50 # 1 percent activity at max concentrationFloat%
13mutant luminescence IC50 # 1 LU at 0.00305 microMFloat
14mutant luminescence IC50 # 1 at 0.006104 microM (0.006104μM**)Float
15mutant luminescence IC50 # 1 at 0.0122 microM (0.0122μM**)Float
16mutant luminescence IC50 # 1 at 0.0244 microM (0.0244μM**)Float
17mutant luminescence IC50 # 1 at 0.0488 microM (0.0488μM**)Float
18mutant luminescence IC50 # 1 at 0.0976 microM (0.0976μM**)Float
19mutant luminescence IC50 # 1 at 0.1953 microM (0.1953μM**)Float
20mutant luminescence IC50 # 1 at 0.3906 microM (0.3906μM**)Float
21mutant luminescence IC50 # 1 at 0.78125 microM (0.78125μM**)Float
22mutant luminescence IC50 # 1 at 1.5625 microM (1.5625μM**)Float
23mutant luminescence IC50 # 1 at 3.125 microM (3.125μM**)Float
24mutant luminescence IC50 # 1 at 6.25 microM (6.25μM**)Float
25mutant luminescence IC50 # 1 at 12.5 microM (12.5μM**)Float
26mutant luminescence IC50 # 1 at 25 microM (25μM**)Float
27mutant luminescence IC50 # 1 at 50 microM (50μM**)Float
28mutant luminescence IC50 # 1 at 100 microM (100μM**)Float
29mutant luminescence IC50 # 1 control meanFloat
30mutant luminescence IC50 # 1 control standard deviationFloat
31mutant luminescence IC50 # 1 control percent CVFloat
32mutant luminescence IC50 # 1 blank meanFloat
33mutant luminescence IC50 # 1 blank standard deviationFloat
34mutant luminescence IC50 # 1 blank percent CVFloat
35mutant luminescence IC50 # 1 signal-background ratioFloatratio
36mutant luminescence IC50 # 1 plate Z-factorFloat
37QualifierString
38mutant luminescence IC50 # 2 FloatμM
39mutant luminescence IC50 # 2 Hill slopeFloat
40mutant luminescence IC50 # 2 R-squaredFloat
41mutant luminescence IC50 # 2 min concentrationFloatμM
42mutant luminescence IC50 # 2 percent activity at min concentrationFloat%
43mutant luminescence IC50 # 2 max concentrationFloatμM
44mutant luminescence IC50 # 2 perecnt activity at max concentrationFloat%
45mutant luminescence IC50 # 2 LU at 0.003052 microM (0.003052μM**)Float
46mutant luminescence IC50 # 2 LU at 0.006104 microM (0.006104μM**)Float
47mutant luminescence IC50 # 2 LU at 0.0122 microM (0.0122μM**)Float
48mutant luminescence IC50 # 2 LU at 0.0244 microM (0.0244μM**)Float
49mutant luminescence IC50 # 2 LU at 0.0488 microM (0.0488μM**)Float
50mutant luminescence IC50 # 2 LU at 0.0976 microM (0.0976μM**)Float
51mutant luminescence IC50 # 2 LU at 0.1953 microM (0.1953μM**)Float
52mutant luminescence IC50 # 2 LU at 0.3906 microM (0.3906μM**)Float
53mutant luminescence IC50 # 2 LU at 0.78125 microM (0.78125μM**)Float
54mutant luminescence IC50 # 2 LU at 1.5625 microM (1.5625μM**)Float
55mutant luminescence IC50 # 2 LU at 3.125 microM (3.125μM**)Float
56mutant luminescence IC50 # 2 LU at 6.25 microM (6.25μM**)Float
57mutant luminescence IC50 # 2 LU at 12.5 microM (6.25μM**)Float
58mutant luminescence IC50 # 2 LU at 25 microM (12.5μM**)Float
59mutant luminescence IC50 # 2 LU at 50 microM (50μM**)Float
60mutant luminescence IC50 # 2 LU at 100 microM (100μM**)Float
61mutant luminescence IC50 # 2 control meanFloat
62mutant luminescence IC50 # 2 control standard deviationFloat
63mutant luminescence IC50 # 2 control percent CVFloat%
64mutant luminescence IC50 # 2 blank meanFloat
65mutant luminescence IC50 # 2 blank standard deviationFloat
66mutant luminescence IC50 # 2 blank percent CVFloat%
67mutant luminescence IC50 # 2 signal-background ratioFloatratio
68mutant luminescence IC50 # 2 plate Z-factorFloat
69QualifierString
70mutant luminescence IC50 # 3FloatμM
71mutant luminescence IC50 # 3 Hill slopeFloat
72mutant luminescence IC50 # 3 R-squaredFloat
73mutant luminescence IC50 # 3 min concentrationFloatμM
74mutant luminescence IC50 # 3 percent activity at min concentrationFloat%
75mutant luminescence IC50 # 3 max concentrationFloatμM
76mutant luminescence IC50 # 3 percent activity at max concentrationFloat%
77mutant luminescence IC50 # 3 LU at 0.003052 microM (0.003052μM**)Float
78mutant luminescence IC50 # 3 LU at 0.006104 microM (0.006104μM**)Float
79mutant luminescence IC50 # 3 LU at 0.0122 microM (0.0122μM**)Float
80mutant luminescence IC50 # 3 LU at 0.0244 microM (0.0244μM**)Float
81mutant luminescence IC50 # 3 LU at 0.0488 microM (0.0488μM**)Float
82mutant luminescence IC50 # 3 LU at 0.0976 microM (0.0976μM**)Float
83mutant luminescence IC50 # 3 LU at 0.1953 microM (0.1953μM**)Float
84mutant luminescence IC50 # 3 LU at 0.3906 microM (0.3906μM**)Float
85mutant luminescence IC50 # 3 LU at 0.78125 microM (0.78125μM**)Float
86mutant luminescence IC50 # 3 LU at 1.5625 microM (1.5625μM**)Float
87mutant luminescence IC50 # 3 LU at 3.125 microM (3.125μM**)Float
88mutant luminescence IC50 # 3 LU at 6.25 microM (6.25μM**)Float
89mutant luminescence IC50 # 3 LU at 12.5 microM (12.5μM**)Float
90mutant luminescence IC50 # 3 LU at 25 microM (25μM**)Float
91mutant luminescence IC50 # 3 LU at 50 microM (50μM**)Float
92mutant luminescence IC50 # 3 LU at 100 microM (100μM**)Float
93mutant luminescence IC50 # 3 blank meanFloat
94mutant luminescence IC50 # 3 blank standard deviationFloat
95mutant luminescence IC50 # 3 blank percent CVFloat%
96mutant luminescence IC50 # 3 control meanFloat
97mutant luminescence IC50 # 3 control standard deviationFloat
98mutant luminescence IC50 # 3 control percent CVFloat%
99mutant luminescence IC50 # 3 signal-background ratioFloatratio
100mutant luminescence IC50 # 3 plate Z-factorFloat
101QualifierString
102mutant growth mean IC50FloatμM
103mutant growth standard deviationFloatμM
104QualifierString
105mutant growth IC50 # 1 FloatμM
106mutant growth IC50 # 1 Hill slopeFloat
107mutant growth IC50 # 1 R-squaredFloat
108mutant growth IC50 # 1 min concentrationFloatμM
109mutant growth IC50 # 1 percent activity at min concentrationFloat%
110mutant growth IC50 # 1 max concentrationFloatμM
111mutant growth IC50 # 1 percent activity at max concentrationFloat%
112mutant growth IC50 # 1 control meanFloat
113mutant growth IC50 # 1 control standard deviationFloat
114mutant growth IC50 # 1 control percent CVFloat%
115mutant growth IC50 # 1 blank meanFloat
116mutant growth IC50 # 1 blank standard deviationFloat
117mutant growth IC50 # 1 blank percent CVFloat%
118mutant growth IC50 # 1 signal-background ratioFloatratio
119mutant growth IC50 # 1 plate Z-factorFloat
120QualifierString
121mutant growth IC50 # 2 FloatμM
122mutant growth IC50 # 2 Hill slopeFloat
123mutant growth IC50 # 2 R-squaredFloat
124mutant growth IC50 # 2 min concentrationFloatμM
125mutant growth IC50 # 2 percent activity at min concentrationFloat%
126mutant growth IC50 # 2 max concentrationFloatμM
127mutant growth IC50 # 2 percent activity at max concentrationFloat%
128mutant growth IC50 # 2 control meanFloat
129mutant growth IC50 # 2 control standard deviationFloat
130mutant growth IC50 # 2 control percent CVFloat%
131mutant growth IC50 # 2 blank meanFloat
132mutant growth IC50 # 2 blank standard deviationFloat
133mutant growth IC50 # 2 blank percent CVFloat%
134mutant growth IC50 # 2 signal-background ratioFloatratio
135mutant growth IC50 # 2 plate Z-factorFloat
136QualifierString
137mutant growth IC50 # 3 FloatμM
138mutant growth IC50 # 3 Hill slopeFloat
139mutant growth IC50 # 3 R-squaredFloat
140mutant growth IC50 # 3 min concentrationFloatμM
141mutant growth IC50 # 3 percent activity at min concentrationFloat%
142mutant growth IC50 # 3 max concentrationFloatμM
143mutant growth IC50 # 3 percent activity at max concentrationFloat%
144mutant growth IC50 # 3 control meanFloat
145mutant growth IC50 # 3 control standard deviationFloat
146mutant growth IC50 # 3 control percent CVFloat%
147mutant growth IC50 # 3 blank meanFloat
148mutant growth IC50 # 3 blank standard deviationFloat
149mutant growth IC50 # 3 blank percent CVFloat%
150mutant growth IC50 # 3 signal-background ratioFloatratio
151mutant growth IC50 # 3 plate Z-factorFloat
152QualifierString
153WT luminescence mean IC50FloatμM
154WT luminescence standard deviationFloatμM
155QualifierString
156WT luminescence IC50 # 1 FloatμM
157WT luminescence IC50 # 1 Hill slopeFloat
158WT luminescence IC50 # 1 R-squaredFloat
159WT luminescence IC50 # 1 min concentrationFloatμM
160WT luminescence IC50 # 1 percent activity at min concentrationFloat%
161WT luminescence IC50 # 1 max concentrationFloatμM
162WT luminescence IC50 # 1 percentt activity at max concentrationFloat%
163WT luminescence IC50 # 1 control meanFloat
164WT luminescence IC50 # 1 control standard deviationFloat
165WT luminescence IC50 # 1 control percent CVFloat%
166WT luminescence IC50 # 1 blank meanFloat
167WT luminescence IC50 # 1 blank standard deviationFloat
168WT luminescence IC50 # 1 blank percent CVFloat%
169WT luminescence IC50 # 1 signal-background ratioFloatratio
170WT luminescence IC50 # 1 plate Z-factorFloat
171QualifierString
172WT luminescence IC50 # 2 FloatμM
173WT luminescence IC50 # 2 Hill slopeFloat
174WT luminescence IC50 # 2 R-squaredFloat
175WT luminescence IC50 # 2 min concentrationFloatμM
176WT luminescence IC50 # 2 percent activity at min concentrationFloat%
177WT luminescence IC50 # 2 max concentrationFloatμM
178WT luminescence IC50 # 2 percent activity at max concentrationFloat%
179WT luminescence IC50 # 2 control meanFloat
180WT luminescence IC50 # 2 control standard deviationFloat
181WT luminescence IC50 # 2 control percent CVFloat%
182WT luminescence IC50 # 2 blank meanFloat
183WT luminescence IC50 # 2 blank standard deviationFloat
184WT luminescence IC50 # 2 blank percent CVFloat%
185WT luminescence IC50 # 2 signal-background ratioFloat
186WT luminescence IC50 # 2 plate Z-factorFloat
187QualifierString
188WT luminescence IC50 # 3 FloatμM
189WT luminescence IC50 # 3 Hill slopeFloat
190WT luminescence IC50 # 3 R-squaredFloat
191WT luminescence IC50 # 3 min concentrationFloatμM
192WT luminescence IC50 # 3 percent activity at min concentrationFloat%
193WT luminescence IC50 # 3 max concentrationFloatμM
194WT luminescence IC50 # 3 percent activity at max concentrationFloat%
195WT luminescence IC50 # 3 control meanFloat
196WT luminescence IC50 # 3 control standard deviationFloat
197WT luminescence IC50 # 3 control percent CVFloat%
198WT luminescence IC50 # 3 blank meanFloat
199WT luminescence IC50 # 3 blank standard deviationFloat
200WT luminescence IC50 # 3 blank percent CVFloat%
201WT luminescence IC50 # 3 signal-background ratioFloatratio
202WT luminescence IC50 # 3 plate Z-factorFloat

* Activity Concentration. ** Test Concentration.

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