AID	Name	Type	Comment
792	eIF2B IC50 against mutant and WT yeast	confirmatory

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 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) [Pavitt GD, Ramaiah KV, Kimball SR, Hinnebusch AG, Genes Dev. 1998, 12, 514-26.] 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.

Materials

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.

HTS 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 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

HTS 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.

Following analysis in ActivityBase, the data was processed using a custom-designed excel-based tool.

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.

Individual scores were calculated as follows:

(1) For mutant luminescence percent inhibition, Score = percent inhibition

(2) For wild-type luminescence percent inhibition > 30, Score = 1.67 x percent inhibition

(3) For wild-type luminescence percent inhibition < 30, Score = 0

(4) For mutant growth percent inhibition > 30, Score = 1.67 x percent inhibition

(5) For mutant growth percent inhibition < 30, Score = 0

The individual scores were then combined to give an overall score as follows:

SCORE = (Mutant percent inhibition score) - (wild-type percent inhibition score) - (growth percent inhibition score)

Negative scores were replaced with a score of 0.


Activity Outcome

All compounds giving an overall score > 50 are reported as active

Active:
Percent inhibition > 50 in mutant luminescence
Percent inhibition < 30 in wild-type luminescence
Percent inhibition < 30 in mutant growth

All compounds giving an overall score < 50 are reported as inactive

Inactive:
(1) Percent inhibition < 50 in mutant luminescence
(2) Percent inhibition > 50 in mutant luminescence
Percent inhibition > 30 in wild-type luminescence
(3) Percent inhibition > 50 in mutant luminescence
Percent inhibition < 30 in wild-type luminescence
Percent inhibition > 30 in mutant growth

A few compounds had a large difference (>25) between the percent inhibition of mutant and wild-type luminescence. These compounds are reported as inconclusive.

Inconclusive:
Percent inhibition > 50 in mutant luminescence
Percent inhibition > 30 in wild-type luminescence
But difference (percent inhibition mutant - percent inhibition wild-type) > 25

Active and inconclusive compounds will be tested further in dose-response confirmation assays. Results of these tests will be reported later.

Analysis of screening results

Results of the HTS were as follows:

Hits (> 50% luminescence inhibition of mutant, < 30% luminescence inhibition of wild type, < 30% growth inhibition of mutant) = 248
Inconclusive (difference between mutant and wild type luminescence % inhibition > 25) = 9
Inactive = 26940


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)