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BioAssay: AID 651696

Cell Proliferation Assay against a hMSC Cell Line

In recent years, using advanced gene expression profiling techniques the laboratory of Dr. Louis Staudt at NCI/NIH has revealed that a class of lymphoma referred to as Diffuse Large B-Cell Lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond differently to therapies. The sub-group classified as activated B cell-like diffuse large more ..
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 Tested Compounds
 Tested Compounds
All(459)
 
 
Inactive(459)
 
 
 Tested Substances
 Tested Substances
All(461)
 
 
Inactive(461)
 
 
 Related BioAssays
 Related BioAssays
AID: 651696
Data Source: NCGC (CELLPROLIF-HMSC)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-24
Hold-until Date: 2013-01-01
Modify Date: 2013-01-03

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
624476Cytotoxicity counterscreen for NFkB agonists and antagonistsconfirmatory
624479qHTS Assay for Identification of Small Molecule Antagonists for NFkB Signaling Pathway.confirmatory
624478qHTS Assay for Identification of Small Molecule Agonists for NFkB Signaling Pathway.confirmatory
651645Cell Proliferation Assay against the TMD8 Cell Lineconfirmatory
651646Cell Proliferation Assay against the HBL1 Cell Lineconfirmatory
Description:
In recent years, using advanced gene expression profiling techniques the laboratory of Dr. Louis Staudt at NCI/NIH has revealed that a class of lymphoma referred to as Diffuse Large B-Cell Lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond differently to therapies. The sub-group classified as activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) demonstrates activation of B-cell receptors and the NFkappaB signaling pathway. One class of targeted therapeutics showing early clinical success in killing of ABC DLBCL lines are small molecule inhibitors of the Bruton tyrosine kinase (BTK) 11. Thus, we chose to investigate what additional compounds, if any, would synergize with the BTK inhibitor Ibrutinib (PCI-32765). Prior to any combination assays, using an NCATS developed oncology compound library called the Mechanism of Interrogation Plate (MIPE), two well studied ABC DLBCL lines (TMD8 and HBL1) were screened using a cell viability assay (CellTiterGlo) and a 12-point titration series for each single agent. These initial experiments offered insight into the concentration range that offered the best coverage of activity and non-activity to assure relevant data is generated. In addition to the viability signature associated with this library, we examined the apoptotic response of each agent versus the TMD8 line as judged by caspase 3/7 activation. Activity was assayed at both 8 and 16 hour time-points to gain an appreciation of which agents induced an apoptotic response in a reasonable timeframe from a therapeutic exposure perspective. MIPE was also assayed for single agent viability in a human mesenchymal stem cell (hMSC) line as a surrogate of each library member's potential therapeutic index.
Protocol
A total of 500 log growing hMSC cells are seeded per well in 5 uL of a RPMI with glutamine (or Glutamax), without phenol red plus 5% FBS and 1X Pen/Strep into 1536 -solid white high base tissue culture treated Greiner One Bio Plates (789173-F) using a multidrop combi dispenser and a small sterile cassette. Compounds and controls totaling 23nL per well (negative control DMSO and positive control 9.2 uM Bortezomib-final) are immediately added to the plates using a 1536 head pin tool from Kalypsys. The plates are then covered with stainless steel gasket lids from Kalypsys and incubated for 48 hours at 37C under 95% relative humidity with 5% CO2. After 48 hours, the plates are removed and allowed to reach room temperature before 3 uL of Cell Titer Glo reagent (Promega) is added using an Aurora Flying Reagent Dispenser Bioraptor. The plates are then spun at 1000 rpms to remove bubbles and incubated for 15 minutes at room temperature before read on a ViewLux using a luminescent filter with a 10 second exposure. The relative luciferase units are used to calculated percent activity using DMSO as 100% and Bortezomib as 0%.
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Actives compounds represent inhibitors of cell proliferation and inactive compounds are considered to have no (or negligable) effect on proliferation
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Activity_ScoreActivity score.Integer
6Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0003926931 uM (0.000392693μM**)% Activity at given concentration.Float%
16Activity at 0.0007804147 uM (0.000780415μM**)% Activity at given concentration.Float%
17Activity at 0.00118 uM (0.00117806μM**)% Activity at given concentration.Float%
18Activity at 0.00232 uM (0.00231672μM**)% Activity at given concentration.Float%
19Activity at 0.00353 uM (0.00353418μM**)% Activity at given concentration.Float%
20Activity at 0.00468 uM (0.00468253μM**)% Activity at given concentration.Float%
21Activity at 0.00703 uM (0.00703465μM**)% Activity at given concentration.Float%
22Activity at 0.014 uM (0.0138793μM**)% Activity at given concentration.Float%
23Activity at 0.021 uM (0.021085μM**)% Activity at given concentration.Float%
24Activity at 0.041 uM (0.0408261μM**)% Activity at given concentration.Float%
25Activity at 0.063 uM (0.0632551μM**)% Activity at given concentration.Float%
26Activity at 0.122 uM (0.122478μM**)% Activity at given concentration.Float%
27Activity at 0.190 uM (0.189642μM**)% Activity at given concentration.Float%
28Activity at 0.286 uM (0.286268μM**)% Activity at given concentration.Float%
29Activity at 0.563 uM (0.562965μM**)% Activity at given concentration.Float%
30Activity at 0.859 uM (0.858804μM**)% Activity at given concentration.Float%
31Activity at 1.138 uM (1.13785μM**)% Activity at given concentration.Float%
32Activity at 1.709 uM (1.70942μM**)% Activity at given concentration.Float%
33Activity at 3.373 uM (3.37266μM**)% Activity at given concentration.Float%
34Activity at 5.124 uM (5.12366μM**)% Activity at given concentration.Float%
35Activity at 9.921 uM (9.92074μM**)% Activity at given concentration.Float%
36Activity at 15.37 uM (15.371μM**)% Activity at given concentration.Float%
37Activity at 29.76 uM (29.7622μM**)% Activity at given concentration.Float%
38Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
39Activity at 92.17 uM (92.1659μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084681 U54

Data Table (Concise)
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