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BioAssay: AID 449763

uHTS identification of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assay

Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding more ..
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Active(1124)
 
 
Inactive(330552)
 
 
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 Tested Substances
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Active(1125)
 
 
Inactive(330635)
 
 
 Related BioAssays
 Related BioAssays
AID: 449763
Data Source: Burnham Center for Chemical Genomics (BCCG-A399-UPR-CHOP-PrimaryAgonist-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-07-22
Modify Date: 2011-01-14

Data Table ( Complete ):           Active    All
BioActive Compounds: 1124
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
463112Single concentration confirmation of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assayscreening
588556SAR analysis of cytotoxicity in CHO-CHOP cells after 24 hours of exposure to small molecule activators of the apoptotic arm of the Unfolded Protein Responseconfirmatory
602416SAR analysis counterscreen of small molecule activators of the apoptotic arm of the Unfolded Protein Response via a luminescent-based reporter assay - Set 3confirmatory
540312SAR analysis of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assayconfirmatory
588594SAR analysis of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assay - Set 2confirmatory
489024Dose response confirmation of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assayconfirmatory
602434SAR analysis of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assay - Set 3confirmatory
588582SAR analysis counterscreen of small molecule activators of the apoptotic arm of the Unfolded Protein Response via a luminescent-based reporter assay - Set 2confirmatory
449771Summary assay for the identification of small molecule activators of the apoptotic arm of the Unfolded Protein responsesummary2
540305SAR analysis counterscreen of small molecule activators of the apoptotic arm of the Unfolded Protein Response via a luminescent-based reporter assayconfirmatory
588558SAR analysis of cytotoxicity in CHO-XBP1 cells after 24 hours of exposure to small molecule activators of the apoptotic arm of the Unfolded Protein responseconfirmatory
489040Dose response counterscreen of small molecule activators of the apoptotic arm of the Unfolded Protein Response via a luminescent-based reporter assayconfirmatory
588570SAR analysis of cytotoxicity in CHO-XBP1 cells following 6 hours of exposure to small molecule activators of the apoptotic arm of the Unfolded Protein responseconfirmatory
588557SAR analysis of cytotoxicity in the CHO-CHOP cells after 6 hours of exposure to small molecule activators of the apoptotic arm of the Unfolded Protein responseconfirmatory
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1 R03 MH089782-01
Assay Provider: Dr. Randal J Kaufman, University of Michigan

Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding defects or impaired transport from the endoplasmic reticulum (ER). Very recently it has come to light that deregulation of protein synthesis may be a key component in the pathogenesis of cancer and metastasis. When misfolded protein accumulates in the ER lumen, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins and restore homeostatic protein processing. When a stress is prolonged or robust the UPR employs a genetic pathway that results in cell death. The proposed studies are to focus attention to identify and characterize drug-like small molecule activators of the apoptotic pathway
We will investigate the hypotheses that specific and robust activation of the PERK-eIF2a-CHOP (apoptotic) arm will exacerbate the UPR and lead to cell death in malignant cells with hyper-active secretory machinery. This aim is predicated on the hypothesis that the adaptive arm of the UPR can be pharmacologically overwhelmed and lead to a productive CHOP-mediated apoptotic response in human cancer cells.

For this primary screen, a cell-based assay was utilized using a cell line stably transfected to report on the PERK-elF2a-CHOP pathway via a luciferase-based reporter system. Activation of the CHOP-mediated apoptotic response leads to the activation of luciferase expression which can be quantified via luminescence.
Protocol
UPR CHO-CHOP 1536-Well Assay Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect activators of the PERK-eIF2a-CHOP (apoptotic) arm of the Unfolded Protein Response pathway.

B. Materials:
CHO-CHOP Cell Line
F12 nutrient mix HAMs (Invitrogen, Cat# 11765047)
Fetal Bovine Serum, heat-inactivated (Hyclone, Cat# SH30396)
Penicillin/Streptomycin, liquid (Invitrogen, Cat# 15140122)
L-glutamine (100X ) (Invitrogen, Cat# 25030081)
MEM Non-Essential Amino Acids Solution 10 mM (100X) (Invitrogen, Cat# 11140050)
Trypsin-EDTA 0.25% (Invitrogen, Cat# 25200-056)
DPBS (Hyclone, Cat# 30028.02)
T225 TC Flask (Nunc, Cat# 159934)
Cell strainer, 40 um (BD, Cat# 352340)
Aurora 1536 well white solid bottom TC plate (Aurora Biotechnology #00029846)
Tunicamycin (Sigma-Aldrich, Cat# T7765)
Steady-Glo# Luciferase Assay System (1L) (Promega, Cat# E2550)

C. Plate Map:
Positive control (P) in columns 1 and 2, 10ug/mL Tunicamycin
Negative control (N) in columns 3 and 4, No Tunicamycin
Test compound (T) in columns 5 - 48, No Tunicamycin

D. Procedures:
Day1 -Cell Seeding
1. Prepare cell suspensions as described in section F. Cell culture.
2. Set up Kalypsys dispenser as described in section G. Kalypsys dispenser setting.
3. Plate 500 cells/well in 5 uL of assay media into columns 1-48 of a 1536-well assay plate, using straight tip dispense on a Kalypsys dispenser.
4. Centrifuge plates at 500 rpm for 1 minute on Eppendorf centrifuge 5810. Use Kalypsys metal lids.
5. Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2 for 16-18 hours.

Day2 - Compound Addition
6. Using LabCyte Echo, transfer 25 nL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 10 uM, 0.5% DMSO), 25 nL of 2 mg/mL Tunicamycin in DMSO to positive control wells in Columns 1 and 2, and 25 nL of DMSO to negative control wells in Columns 3 and 4.
7. Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge.
8. Incubate plates in incubator (37 degrees, 100% relative humidity, 5% CO2) for 6 hours.
9. Set up Kalypsys dispenser and Perkin Elmer Envision as described in section G. Kalypsys dispenser setting and H. Envision setting.
10. Following 6 hours incubation, remove lid and incubate plate for 10 min in at room temp.
11. Add 3 uL of Steady-Glo to each wells (Columns 1 - 48) using a Kalypsys dispenser.
12. Centrifuge plates at 2000 rpm for 2 minutes on a Vspin centrifuge.
13. Lid plate and incubate for 10 min at room temp.
14. Read plates using Envision using a luminescence protocol.

E. Recipe:
Growth Media
F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin, 1X L-glutamine, and 1X MEM-NEAA
Assay Media
Filtered Growth Media
Trypsin
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Positive Control
Tunicamycin at 10 ug/mL final.
(Note: Tunicamycin is reconstituted in DMSO to a concentration of 10mg/ml).

F. Cell Culture:
Prepare Growth Media/Assay Media as described in section E. Recipe.
Procedure to expand and maintain cells:
CHO-CHOP cells are seeded into T225 flasks at 3.75 x 105 cells. Cells are passaged twice a week (Monday or Tuesday, and Thursday or Friday depending on cell growth). Confluency should be maintained at <75%. After 3 days incubation, ~2.5X107 cells are expected per T225 flask. Incubate cells in 5% CO2.
1. Put media in water bath and leave for 30 minutes. Also leave Trypsin at room temp for 15 minutes. Keep DPBS at room temp (no need to warm up DPBS at 37 degrees).
2. Aspirate off old media from T225 flask.
3. Wash the flasks with 20 mL DPBS per T225 flask. Leave cells in DPBS for about 30 seconds.
4. Add 6.5 mL 0.05% Trypsin solution into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 900 rpm for 5 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh growth media with p1000 pipette.
9. Add 19 mL of growth media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and prepare stock flasks with
3.00 x 105 cells per T225 flask for 4 days incubation.
3.75 x 105 cells per T225 flask for 3 days incubation.
Procedure to prepare cells for the assay:
Follow steps 1 - 3 above
4. Add 5 mL Trypsin into the flask. Rock the flask gently so that the solution covers all over the surface.
5. Allow the cells to detach by incubating at room temperature for about 4 minutes.
6. Wash the flask with 25 mL fresh growth media.
7. Collect the cell suspension in a 50 mL sterile conical tube.
8. Centrifuge at 500 rpm for 10 minutes. Once pelleted, remove the supernatant and resuspend the cell pellet carefully in 1 mL fresh assay with p1000 pipette.
9. Add 19 mL of assay media and mix gently.
10. Filter the cell suspension with cell strainer.
11. Count the cells and adjust cell density to 1.0x105 cells/mL in media.
Comment
Compounds that test with activity greater than or equal to 40% activation at 10 uM concentration relative to the positive control are defined as actives in the primary assay.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 10 uM concentration:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 10 uM (10μM**)% Activity in primary screeningFloat%
2Value (10μM**)Observed luminescence value of the compoundFloatRLU
3Mean HighMean Luminescence ratio of positive controls in the corresponding plateFloatRLU
4STD Deviation HighStandard deviation (n=64) of positive controls in the corresponding plateFloatRLU
5Mean LowMean Luminescence ratio of negative controls in the corresponding plateFloatRLU
6STD Deviation LowStandard deviation (n=64) of negative controls in the corresponding plateFloatRLU

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH089782-01

Data Table (Concise)
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