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BioAssay: AID 588570

SAR analysis of cytotoxicity in CHO-XBP1 cells following 6 hours of exposure to small molecule activators of the apoptotic arm of the Unfolded Protein response

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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AID: 588570
Data Source: Burnham Center for Chemical Genomics (SBCCG-A728-UPR-CHOP-DryPowder-6H-cytotox-in-XBP1-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-19
Hold-until Date: 2012-10-01
Modify Date: 2012-10-01

Data Table ( Complete ):           Active    All
BioActive Compounds: 12
Depositor Specified Assays
AIDNameTypeComment
449763uHTS identification of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assayscreeningPrimary Screen
449771Summary assay for the identification of small molecule activators of the apoptotic arm of the Unfolded Protein responsesummarySummary AID.
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, Sanford-Burnham Medical Research Institute (SBMRI, San Diego)

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 adaptive arm of 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 adaptive pathway. We will investigate the hypotheses that activation of the IRE1-XBP1 (adaptive) arm will facilitate a prolonged recovery opportunity in pathologies where cells are burdened with an accumulation of mis-folded or poorly secreted proteins.

For the primary screen, a cell-based assay was utilized using a cell line stably transfected to report on the IRE1-XBP1 pathway via a luciferase-based reporter system. Activation of the IRE1-XBP1-mediated adaptive response leads to the activation of luciferase expression which can be quantified via luminescence.


The goal of this assay is to test the cytotoxicity after 6 hours of exposure to SAR compounds derived from hits from "uHTS identification of small molecule activators of the apoptotic arm of the Unfolded Protein response via a luminescent-based reporter assay", AID 449763. Compounds are either acquired from commercial sources or synthesized internally.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to test the dose dependent cytotoxic response after 6 hours of exposure to activators of the PERK-eIF2a-CHOP (apoptotic) arm of the Unfolded Protein Response pathway in the CHO-XBP1 cell line

B. Materials:
CHO-XBP1 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)
ATP Lite 1-Step (PerkinElmer, Cat# 6016739)

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 1000 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 varying volumes of test compounds in DMSO to achieve appropriate dose concentrations and range, and backfill with DMSO to equilibrate DMSO concentrations in all wells (Col. 5 - 48). Add equal volume of DMSO to negative control wells (Col. 3 - 4) and tunicamycin in DMSO to positive control wells (Col. 1 - 2) to achieve a final assay concentration of tunicamycin of 10 ug/mL. Final DMSO concentration should not exceed 1%.
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 ATPLite 1 Step, prepared according to the manufacturer's recommendation, 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-XBP1 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 1.0 x 105 cells/mL in media.
Comment
Compounds with and IC50_Mean <= 20 uM are defined as actives in this 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 and is not applicable in this assay.

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
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:

QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]

This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is

Score = 82 + 3*(pIC50 - 3)*QC,

where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 80 uM_first_point (80μM**)% Activity at the test concentrationFloat%
9% Activity at 40 uM_first_point (40μM**)% Activity at the test concentrationFloat%
10% Activity at 20 uM_first_point (20μM**)% Activity at the test concentrationFloat%
11% Activity at 10 uM_first_point (10μM**)% Activity at the test concentrationFloat%
12% Activity at 5 uM_first_point (5μM**)% Activity at the test concentrationFloat%
13% Activity at 2.5 uM_first_point (2.5μM**)% Activity at the test concentrationFloat%
14% Activity at 1.25 uM_first_point (1.25μM**)% Activity at the test concentrationFloat%
15% Activity at 0.625 uM_first_point (0.625μM**)% Activity at the test concentrationFloat%
16% Activity at 0.3125 uM_first_point (0.3125μM**)% Activity at the test concentrationFloat%
17% Activity at 0.15625 uM_first_point (0.15625μM**)% Activity at the test concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 80 uM_second_point (80μM**)% Activity at the test concentrationFloat%
20% Activity at 40 uM_second_point (40μM**)% Activity at the test concentrationFloat%
21% Activity at 20 uM_second_point (20μM**)% Activity at the test concentrationFloat%
22% Activity at 10 uM_second_point (10μM**)% Activity at the test concentrationFloat%
23% Activity at 5 uM_second_point (5μM**)% Activity at the test concentrationFloat%
24% Activity at 2.5 uM_second_point (2.5μM**)% Activity at the test concentrationFloat%
25% Activity at 1.25 uM_second_point (1.25μM**)% Activity at the test concentrationFloat%
26% Activity at 0.625 uM_second_point (0.625μM**)% Activity at the test concentrationFloat%
27% Activity at 0.3125 uM_second_point (0.3125μM**)% Activity at the test concentrationFloat%
28% Activity at 0.15625 uM_second_point (0.15625μM**)% Activity at the test concentrationFloat%
29IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2IC50 value determined using a sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2Standard Error of the IC50 valueFloatμM
32nH_2Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 80 uM_third_point (80μM**)% Activity at the test concentrationFloat%
35% Activity at 40 uM_third_point (40μM**)% Activity at the test concentrationFloat%
36% Activity at 20 uM_third_point (20μM**)% Activity at the test concentrationFloat%
37% Activity at 10 uM_third_point (10μM**)% Activity at the test concentrationFloat%
38% Activity at 5 uM_third_point (5μM**)% Activity at the test concentrationFloat%
39% Activity at 2.5 uM_third_point (2.5μM**)% Activity at the test concentrationFloat%
40% Activity at 1.25 uM_third_point (1.25μM**)% Activity at the test concentrationFloat%
41% Activity at 0.625 uM_third_point (0.625μM**)% Activity at the test concentrationFloat%
42% Activity at 0.3125 uM_third_point (0.3125μM**)% Activity at the test concentrationFloat%
43% Activity at 0.15625 uM_third_point (0.15625μM**)% Activity at the test concentrationFloat%
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 80 uM_fourth_point (80μM**)% Activity at the test concentrationFloat%
46% Activity at 40 uM_fourth_point (40μM**)% Activity at the test concentrationFloat%
47% Activity at 20 uM_fourth_point (20μM**)% Activity at the test concentrationFloat%
48% Activity at 10 uM_fourth_point (10μM**)% Activity at the test concentrationFloat%
49% Activity at 5 uM_fourth_point (5μM**)% Activity at the test concentrationFloat%
50% Activity at 2.5 uM_fourth_point (2.5μM**)% Activity at the test concentrationFloat%
51% Activity at 1.25 uM_fourth_point (1.25μM**)% Activity at the test concentrationFloat%
52% Activity at 0.625 uM_fourth_point (0.625μM**)% Activity at the test concentrationFloat%
53% Activity at 0.3125 uM_fourth_point (0.3125μM**)% Activity at the test concentrationFloat%
54% Activity at 0.15625 uM_fourth_point (0.15625μM**)% Activity at the test concentrationFloat%
55IC50_Qualifier_3This qualifier is to be used with the next TID, IC50_3. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
56IC50_3IC50 value determined using a sigmoidal dose response equationFloatμM
57Std.Err(IC50)_3Standard Error of the IC50 valueFloatμM
58nH_3Hill coefficient determined using sigmoidal dose response equationFloat
59Excluded_Points_fifth_pointFlags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
60% Activity at 80 uM_fifth_point (80μM**)% Activity at the test concentrationFloat%
61% Activity at 40 uM_fifth_point (40μM**)% Activity at the test concentrationFloat%
62% Activity at 20 uM_fifth_point (20μM**)% Activity at the test concentrationFloat%
63% Activity at 10 uM_fifth_point (10μM**)% Activity at the test concentrationFloat%
64% Activity at 5 uM_fifth_point (5μM**)% Activity at the test concentrationFloat%
65% Activity at 2.5 uM_fifth_point (2.5μM**)% Activity at the test concentrationFloat%
66% Activity at 1.25 uM_fifth_point (1.25μM**)% Activity at the test concentrationFloat%
67% Activity at 0.625 uM_fifth_point (0.625μM**)% Activity at the test concentrationFloat%
68% Activity at 0.3125 uM_fifth_point (0.3125μM**)% Activity at the test concentrationFloat%
69% Activity at 0.15625 uM_fifth_point (0.15625μM**)% Activity at the test concentrationFloat%
70Excluded_Points_sixth_pointFlags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
71% Activity at 80 uM_sixth_point (80μM**)% Activity at the test concentrationFloat%
72% Activity at 40 uM_sixth_point (40μM**)% Activity at the test concentrationFloat%
73% Activity at 20 uM_sixth_point (20μM**)% Activity at the test concentrationFloat%
74% Activity at 10 uM_sixth_point (10μM**)% Activity at the test concentrationFloat%
75% Activity at 5 uM_sixth_point (5μM**)% Activity at the test concentrationFloat%
76% Activity at 2.5 uM_sixth_point (2.5μM**)% Activity at the test concentrationFloat%
77% Activity at 1.25 uM_sixth_point (1.25μM**)% Activity at the test concentrationFloat%
78% Activity at 0.625 uM_sixth_point (0.625μM**)% Activity at the test concentrationFloat%
79% Activity at 0.3125 uM_sixth_point (0.3125μM**)% Activity at the test concentrationFloat%
80% Activity at 0.15625 uM_sixth_point (0.15625μM**)% Activity at the test concentrationFloat%
81IC50_Qualifier_4This qualifier is to be used with the next TID, IC50_4. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
82IC50_4IC50 value determined using a sigmoidal dose response equationFloatμM
83Std.Err(IC50)_4Standard Error of the IC50 valueFloatμM
84nH_4Hill coefficient determined using sigmoidal dose response equationFloat
85Excluded_Points_seventh_pointFlags to indicate which of the seventh dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
86% Activity at 80 uM_seventh_point (80μM**)% Activity at the test concentrationFloat%
87% Activity at 40 uM_seventh_point (40μM**)% Activity at the test concentrationFloat%
88% Activity at 20 uM_seventh_point (20μM**)% Activity at the test concentrationFloat%
89% Activity at 10 uM_seventh_point (10μM**)% Activity at the test concentrationFloat%
90% Activity at 5 uM_seventh_point (5μM**)% Activity at the test concentrationFloat%
91% Activity at 2.5 uM_seventh_point (2.5μM**)% Activity at the test concentrationFloat%
92% Activity at 1.25 uM_seventh_point (1.25μM**)% Activity at the test concentrationFloat%
93% Activity at 0.625 uM_seventh_point (0.625μM**)% Activity at the test concentrationFloat%
94% Activity at 0.3125 uM_seventh_point (0.3125μM**)% Activity at the test concentrationFloat%
95% Activity at 0.15625 uM_seventh_point (0.15625μM**)% Activity at the test concentrationFloat%
96Excluded_Points_eighth_pointFlags to indicate which of the eighth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
97% Activity at 80 uM_eighth_point (80μM**)% Activity at the test concentrationFloat%
98% Activity at 40 uM_eighth_point (40μM**)% Activity at the test concentrationFloat%
99% Activity at 20 uM_eighth_point (20μM**)% Activity at the test concentrationFloat%
100% Activity at 10 uM_eighth_point (10μM**)% Activity at the test concentrationFloat%
101% Activity at 5 uM_eighth_point (5μM**)% Activity at the test concentrationFloat%
102% Activity at 2.5 uM_eighth_point (2.5μM**)% Activity at the test concentrationFloat%
103% Activity at 1.25 uM_eighth_point (1.25μM**)% Activity at the test concentrationFloat%
104% Activity at 0.625 uM_eighth_point (0.625μM**)% Activity at the test concentrationFloat%
105% Activity at 0.3125 uM_eighth_point (0.3125μM**)% Activity at the test concentrationFloat%
106% Activity at 0.15625 uM_eighth_point (0.15625μM**)% Activity at the test concentrationFloat%

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

Data Table (Concise)
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