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BioAssay: AID 540303

qHTS for Inhibitors of Cell Surface uPA Generation

The overall objective of this project is to perform a quantitative high throughput screen (qHTS) to identify novel compounds that inhibit the generation of cell surface urokinase activity. The urokinase plasminogen activator (uPA) is one of the two primary endogenous systems that mediate plasminogen activation into plasmin. Members of this system, including uPA and its receptor (uPAR), are more ..
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 Tested Compounds
 Tested Compounds
All(325237)
 
 
Active(1021)
 
 
Inactive(313706)
 
 
Inconclusive(10523)
 
 
 Tested Substances
 Tested Substances
All(325463)
 
 
Active(1021)
 
 
Inactive(313911)
 
 
Inconclusive(10531)
 
 
AID: 540303
Data Source: NCGC (uPA001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-07-27

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 1021
Related Experiments
AIDNameTypeComment
493178qHTS for Inhibitors of Cell Surface uPA Generation: Assay SummarySummarydepositor-specified cross reference: Summary of project
720517qHTS for Inhibitors of Cell Surface uPA Generation: FP59 Counterscreen for Cherry-picked CompoundsConfirmatorydepositor-specified cross reference
720518qHTS for Inhibitors of Cell Surface uPA Generation: Wild-type Protective Antigen (WT-PrAg) Counterscreen for Cherry-picked CompoundsConfirmatorydepositor-specified cross reference
720519qHTS for Inhibitors of Cell Surface uPA Generation: Confirmatory Assay for Cherry-picked CompoundsConfirmatorydepositor-specified cross reference
493164qHTS for Inhibitors of Cell Surface uPA Generation: Validation AssayConfirmatorysame project related to Summary assay
Description:
The overall objective of this project is to perform a quantitative high throughput screen (qHTS) to identify novel compounds that inhibit the generation of cell surface urokinase activity. The urokinase plasminogen activator (uPA) is one of the two primary endogenous systems that mediate plasminogen activation into plasmin. Members of this system, including uPA and its receptor (uPAR), are overexpressed in several malignant tumors. This system plays a major role in adhesion, migration, invasion and metastasis of cancer cells. Small molecule inhibitors of the uPA system will serve as probes to understand the molecular mechanisms of the uPA cascade and better understanding the microenvironmental circumstances that facilitate the activation of plasminogen by uPA, and finally may provide novel candidate agents for anticancer drug therapy.

This uPA assay was developed by modifying the mechanism of anthrax lethal toxin to require activation by uPA. It utilizes the uPA-specific substrate, PrAg-U2, and the detection reagent ATP-Lite. Upon inhibition of the uPA activation cascade, the subsequent cell surface uPA activity-dependent translocation of LF into the cytoplasm is inhibited. ATP-Lite is used to measure intracellular ATP concentrations, which remain increased if LF entry into the cytoplasm is inhibited. This validation assay was performed using a small set of validation compounds.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH086467
Assay Submitter (PI): Thomas Bugge, National Institute of Dental and Craniofacial Research, NIH
Protocol
Cultured RAW 264.7 were dispensed into 1536-well clear bottom, white walled tissue culture plates at a density of 4000 cells/well and were incubated overnight (12-16 h) at 37 C in a 5% CO2 incubator. Serially diluted compounds in 23 nl DMSO were added to cells. This was followed by the addition of 0.5 ul water containing 1 ug/ml PrAg-U2 and 1 ug/ml LF. Compound and reagent-treated plates were incubated for 24 h at 37 C in a 5% CO2 incubator. Perkin Elmer ATPlitetrade mark 1 step (3 ul) was then added to each well. Luminescence was detected using a Perkin Elmer ViewLuxtrade mark plate reader.
1.Dispensing RAW 264.7 cells into 1536-well, white walled tissue culture plates at a density of 4000 cells/well (4 uL).
2.Incubation at 37 C in a 5% CO2 incubator overnight.
3.Serially diluting compounds (1:5 serial dilution; 0.0024 uM to 3.83 uM final concentration) in 23 nl DMSO.
4.Adding of 0.5 ul water containing 1 ug/ml PrAg-U2 and 1 ug/ml LF.
5.Incubating the compound and reagent-treated plates for 24 hours at 37 C in a 5% CO2 incubator.
6.Adding ATP-Lite (3 ul).
7.Reading plates by ViewLux with an exposure length of 5 seconds.
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: RAW 264.7
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
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
5Curve_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
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.307 uM (0.307μM**)% Activity at given concentration.Float%
15Activity at 1.530 uM (1.53μM**)% Activity at given concentration.Float%
16Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
17Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
18Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086467

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