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 ..
Depositor Specified Assays
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.
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.
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 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.
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)
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.
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)
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.
Please direct correspondence to Andrew Napper (firstname.lastname@example.org).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)