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BioAssay: AID 1755

SAR analysis of compounds that potentiate TRAIL-induced apoptosis in MDA-MB-435 cells.

This assay was developed and performed to confirm hits originally identified in "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells" (AID 1443) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally. ..more
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 Tested Compounds
 Tested Compounds
All(9)
 
 
Active(2)
 
 
Inactive(7)
 
 
 Tested Substances
 Tested Substances
All(10)
 
 
Active(3)
 
 
Inactive(7)
 
 
AID: 1755
Data Source: Burnham Center for Chemical Genomics (BCCG-A194-TRAIL-MDA-MB-435-DryPowder-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-05-13
Modify Date: 2011-01-05

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: tumor necrosis factor (ligand) superfamily, member 10 [Homo sapiens]
Description ..   
Protein Family: TNF

Gene:TNFSF10     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
1443uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cellsScreening depositor-specified cross reference
1447Identification of compounds which are cytotoxic to PPC-1 cells.Screening depositor-specified cross reference
1624Confirmation screening of compounds that potentiate TRAIL-induced apoptosis of cancer cells.Confirmatory depositor-specified cross reference
1640Summary of compounds that potentiate TRAIL-induced apoptosis of cancer cells.Summary2 depositor-specified cross reference
1745Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in normal 184B5 cellsOther same project related to Summary assay
1746Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in primary human hepatocytes.Other same project related to Summary assay
1747Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435Other same project related to Summary assay
1748Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MCF7Other same project related to Summary assay
1752SAR analysis of compounds that potentiate TRAIL-induced apoptosis in PPC-1 cells.Confirmatory same project related to Summary assay
1754SAR analysis of compounds cytotoxic to PPC-1 cells.Confirmatory same project related to Summary assay
1756SAR analysis of compounds cytotoxic to MDA-MB-435 cells.Confirmatory same project related to Summary assay
1765Mechanism of action of compounds that potentiate TRAIL-induced apoptosis in PPC-1 cellsOther same project related to Summary assay
1773Comparison of the effects of compounds on TRAIL-mediated extrinsic apoptotic pathway in TRAIL-resistant MDA-MB-435 cells.Other same project related to Summary assay
1774Comparison of the effects of compounds on apoptotic pathways (intrinsic and ER stress pathway) in TRAIL-resistant MDA-MB-435 cells.Other same project related to Summary assay
2619SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435.Other same project related to Summary assay
2647SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435/MDR.Other same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: X01 MH083230-01
Assay Provider: Dr. Dmitri Rozanov, Sanford-Burnham Medical Research Institute, San Diego CA

This assay was developed and performed to confirm hits originally identified in "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells" (AID 1443) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

The TRAIL-resistant cell line, MDA-MB-435 is used, because we would like to determine if compounds can potentiate TRAIL-mediated cytotoxicity not only in TRAIL-sensitive PPC-1 carcinoma cells(AIDs 1443 and 1624) but also in TRAIL-resistant cells.

Cytotoxic chemotherapy induces apoptosis via a pathway involving mitochondria, sometimes referred to as the "intrinsic pathway." An acquired resistance to anticancer drugs commonly results from the accumulation of defects in components of the mitochondrial pathway for apoptosis. Discovering and identifying alternative pathways for triggering tumor cells apoptosis offer hope for more effective outcomes. Members of the Tumor Necrosis Factor (TNF) family of "death receptors" induce apoptosis via a direct mechanism that proceeds without involving mitochondria - referred to as the "extrinsic pathway." These cytokine receptors are frequently employed by immune cells to attack tumors. The PI believes that a successful strategy can be implemented by identifying specific chemicals that will selectively potentiate the therapeutic effects of the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). Unlike other TNF-family members, TRAIL is a powerful and safe cancer therapeutic because it can induce broad spectrum apoptosis of different cancer cells but not normal cells. Unfortunately many cancer cells have proven to be resistant to TRAIL alone.

The goal of this project is to screen for chemical compounds that selectively sensitize tumor cells to the extrinsic apoptosis pathway activated by TRAIL, without affecting other cell death pathways and normal cells. These compounds would provide useful research tools for interrogating mechanisms of TRAIL-resistance, and they also might serve as the basis for future drug development programs to create a new generation of non-toxic anticancer drugs that restore sensitivity to endogenous pathways used by the immune system for eradicating tumors.

References:
Bodmer JL, Meier P, Tschopp J, Schneider P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 2000, 275:20632-7.

Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B, Hillan K, Totpal K, DeForge L, Schow P, Hooley J, Sherwood S, Pai R, Leung S, Khan L, Gliniak B, Bussiere J, Smith CA, Strom SS, Kelley S, Fox JA, Thomas D, Ashkenazi A. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 2001, 7:383-5.

Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003, 63:5390-400

Greil R, Anether G, Johrer K, Tinhofer I. Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools. J Leukoc Biol 2003, 74:311-30.

Smyth MJ, Takeda K, Hayakawa Y, Peschon JJ, van den Brink MR, Yagita H. Nature's TRAIL-on a path to cancer immunotherapy. Immunity 2003, 18:1-6.
Protocol
1) MDA-MB-435 cells and TRAIL were provided by the assay provider
2) ATPLite (Perkin Elmer)
Cytotoxicity assay protocol:
Day 1
1) MDA-MB-435 cells are grown in DMEM, 10% FBS, 4.5 g/l glucose, 2mM L-glutamine, 1mM Na-pyruvate, 15ug/ml gentamycin and are harvested at 80-90% confluency.
2) Cells are then seeded into a 384 well plate (Greiner # 781098 white, clear-bottom, 384, TC-treated plate) at a concentration of 500 cells/well in 45ul and incubated for 24 hours at 37 oC in a CO2 incubator to obtain 10-20% of confluence.
Day 2
1) Add 5 ul of 20X compound in 10% DMSO to wells to obtain 10 uM (final assay concentration of DMSO is 0.5% in 100ul final assay volume per well).
*Drugs are tested in 10-point dilution series with dilution factor 2 starting from 10uM final assay concentration.
2) Add 45uL of Assay Media to each well
3) Incubate cells 4 hours at 37 oC in a CO2 incubator.
4) Add 5 ul of 20X TRAIL in PBS without Mg2+ and Ca2+ (Final assay concentration of TRAIL is 100ng/mL in 100ul final assay volume per well).
*100 ng/mL TRAIL plus 10uM Doxirubicin is used as the 100% cytotoxicity positive control.
5) Incubate cells for an additional 24 hours at 37 oC in a CO2 incubator.
Day 3
1) Add 10 ul of ATPlite reagent to each well and shake plate for 20 min at room temperature.
2) Read luminescence using Envision 384 US Lum (integration time= 0.1s, 0mm from top of plate.)
Comment
Compounds with an IC50 < 10 uM are considered "active."
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 Bir1/2 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 this 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's activatory 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 the XIAP 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 90 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.
A score of 82 is given to active compounds selected from plates:
a) That do not have a Hill coefficient associated with them and have a qualifier of < or >.
or
b) The value of + 3*(pIC50-3)*QC, is < 0.500
Active compounds will have a score >= 82.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: MDA-MB-435
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column;if qualifier is ">", IC50 result is greater than that value. if qualifier is "<", IC50 result is smaller than that value.String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloat
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was (1) excluded and (0) means the point was not excluded, for the titration series going from low to high compound concentrations.String
6% inhibition at 10 uM_first_point (10μM**)% inhibition at a given concentrationFloat%
7% inhibition at 5 uM_first_point (5μM**)% inhibition at a given concentrationFloat%
8% inhibition at 2.5 uM_first_point (2.5μM**)% inhibition at a given concentrationFloat%
9% inhibition at 1.25 uM_first_point (1.25μM**)% inhibition at a given concentrationFloat%
10% inhibition at 0.625 uM_first_point (0.625μM**)% inhibition at a given concentrationFloat%
11% inhibition at 0.3125 uM_first_point (0.3125μM**)% inhibition at a given concentrationFloat%
12% inhibition at 0.15625 uM_first_point (0.15625μM**)% inhibition at a given concentrationFloat%
13% inhibition at 0.078125 uM_first_point (0.078125μM**)% inhibition at a given concentrationFloat%
14% inhibition at 0.0390625 uM_first_point (0.0390625μM**)% inhibition at a given concentrationFloat%
15% inhibition at 0.01953125 uM_first_point (0.0195312μM**)% inhibition at a given concentrationFloat%
16Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was (1) excluded and (0) means the point was not excluded, for the titration series going from low to high compound concentrations.String
17% inhibition at 10 uM_second_point (10μM**)% inhibition at a given concentrationFloat%
18% inhibition at 5 uM_second_point (5μM**)% inhibition at a given concentrationFloat%
19% inhibition at 2.5 uM_second_point (2.5μM**)% inhibition at a given concentrationFloat%
20% inhibition at 1.25 uM_second_point (1.25μM**)% inhibition at a given concentrationFloat%
21% inhibition at 0.625 uM_second_point (0.625μM**)% inhibition at a given concentrationFloat%
22% inhibition at 0.3125 uM_second_point (0.3125μM**)% inhibition at a given concentrationFloat%
23% inhibition at 0.15625 uM_second_point (0.15625μM**)% inhibition at a given concentrationFloat%
24% inhibition at 0.078125 uM_second_point (0.078125μM**)% inhibition at a given concentrationFloat%
25% inhibition at 0.0390625 uM_second_point (0.0390625μM**)% inhibition at a given concentrationFloat%
26% inhibition at 0.01953125 uM_second_point (0.0195312μM**)% inhibition at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: X01 MH083230-01

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