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BioAssay: AID 1770

qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)

Clk4 (Invitrogen, cat# PV3839) was assayed using ATP and the RS repeat peptide (AnaSpec cat # 61722) as substrates. Following the Clk4 reaction, the remaining ATP levels were detected using Promega Kinase-Glo technology wherein the remaining ATP from the kinase reaction is detected using Ultra-Glo luciferase and D-luciferin which generates a bioluminescence signal from the ATP. The positive control for the assay was TG003 (Sigma-Aldrich, cat# 300801-52-9) which has been described as an inhibitor of Clk 4[1]. ..more
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 Tested Compounds
 Tested Compounds
All(1352)
 
 
Active(332)
 
 
Inactive(794)
 
 
Inconclusive(226)
 
 
 Tested Substances
 Tested Substances
All(1352)
 
 
Active(332)
 
 
Inactive(794)
 
 
Inconclusive(226)
 
 
AID: 1770
Data Source: NCGC (CLK835)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-05-18
Modify Date: 2009-05-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 332
Related Experiments
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AIDNameTypeProbeComment
1379Counterscreen for Luciferase (Kinase-Glo TM) InhibitionConfirmatory depositor-specified cross reference
1795qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)Confirmatory depositor-specified cross reference
1969Confirmaiton Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)Confirmatory depositor-specified cross reference
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)Confirmatory depositor-specified cross reference
1983Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)Confirmatory depositor-specified cross reference
1997qHTS for Inhibitors of CDC-like Kinase 4: SummarySummary3 depositor-specified cross reference
2705Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay)Confirmatory depositor-specified cross reference
2710Kinase Inhibition Profile Study on Inhibitors of CDC-like Kinase 4Other depositor-specified cross reference
488872qHTS for Inhibitors of DYRK1A: SummarySummary1 depositor-specified cross reference
488883qHTS for Inhibitors of DYRK1B: SummarySummary1 depositor-specified cross reference
488887qHTS for Inhibitors of CDC-like Kinase 1 and 4: SummarySummary1 depositor-specified cross reference
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2Confirmatory depositor-specified cross reference
493206Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay): round 2 SARConfirmatory depositor-specified cross reference
504421Kinase Inhibition Study on Inhibitors of CDC-like Kinase 4 (Reaction Biology data): SAR round 2Confirmatory depositor-specified cross reference
504424Kinase Inhibition Study on Inhibitors of Dyrk1a (Reaction Biology data)Confirmatory depositor-specified cross reference
504427Kinase Inhibition Study on Inhibitors of CDC-like Kinase 2 (Reaction Biology data)Confirmatory depositor-specified cross reference
504428Kinase Inhibition Study on Inhibitors of CDC-like Kinase 3 (Reaction Biology data)Confirmatory depositor-specified cross reference
504429Kinase Inhibition Study on Inhibitors of Dyrk1b (Reaction Biology data)Confirmatory depositor-specified cross reference
504430Kinase Inhibition Study on Inhibitors of CDC-like Kinase 1 (Reaction Biology data)Confirmatory depositor-specified cross reference
1771qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)Confirmatory same project related to Summary assay
504534Secondary Assay for Inhibitors of Human Cdc-like kinase 4 (Clk4): HEK-293 Cell Viability AssayConfirmatory same project related to Summary assay
624034Reaction Biology data for inhibitors of CLK4: Probe SARConfirmatory same project related to Summary assay
624045Reaction Biology data for inhibitors of DYRK1A: Probe SARConfirmatory same project related to Summary assay
624046Reaction Biology data for inhibitors of DYRK1B: Probe SARConfirmatory same project related to Summary assay
624047Reaction Biology data for inhibitors of CLK1: Probe SARConfirmatory same project related to Summary assay
624048Reaction Biology data for inhibitors of CLK2: Probe SARConfirmatory same project related to Summary assay
624049Reaction Biology data for inhibitors of CLK3: Probe SARConfirmatory same project related to Summary assay
624093Confirmation Assay for Inhibitors of Dyrk1a (Kinase-Glo Assay): KU probeConfirmatory same project related to Summary assay
624094Confirmation Assay for Inhibitors of Clk4 (Kinase-Glo Assay): KU probeConfirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1487qHTS Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1498Confirmation Concentration-Response Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1487qHTS Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1498Confirmation Concentration-Response Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1459Validation of Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1487qHTS Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
1498Confirmation Concentration-Response Assay for Modulators of Lamin A SplicingConfirmatory same project related to Summary assay
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1R03MH084827-01
Assay Submitter (PI): Tom Misteli

NCGC Assay Overview:
Clk4 (Invitrogen, cat# PV3839) was assayed using ATP and the RS repeat peptide (AnaSpec cat # 61722) as substrates. Following the Clk4 reaction, the remaining ATP levels were detected using Promega Kinase-Glo technology wherein the remaining ATP from the kinase reaction is detected using Ultra-Glo luciferase and D-luciferin which generates a bioluminescence signal from the ATP. The positive control for the assay was TG003 (Sigma-Aldrich, cat# 300801-52-9) which has been described as an inhibitor of Clk 4[1].
Protocol
NCGC Assay Protocol Summary:
Two uL/well of substrate-buffer solution (100 uM RS peptide, 1 uM ATP, 25 mM Tris pH7.5, 10 mM MgCl2, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100 final concentration) was dispensed into 1536-well, assay plates (Greiner, solid white medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Using a Kalypsys pin tool equipped with a 1536-pin tool, 23 nL of compound solution was transferred to the assay plate. One uL/ well Clk4-buffer solution (25 nM Clk4, 25 mM Tris pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100, final concentrations) was then added using the FRD yielding a total kinase reaction volume of 3 uL/ well. After 4.5 hours of room temperature incubation, 3 uL Kinase-Glo reagent was added for a final assay volume of 6 uL/ well. Luminescence was detected with the ViewLux plate reader (Perkin Elmer, Waltham, MA) after 5 min incubation using a 5 second exposure time and 2x binning.
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".
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.
3. Compounds that interfere with the Ultra-Glo luciferase could interfere with this assay. PubChem AID: 1379 can be used as counter-screen for this [2].
Categorized Comment - additional comments and annotations
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 0.0001220703 uM (0.00012207μM**)% Activity at given concentration.Float%
15Activity at 0.0002441406 uM (0.000244141μM**)% Activity at given concentration.Float%
16Activity at 0.0004882813 uM (0.000488281μM**)% Activity at given concentration.Float%
17Activity at 0.0009765625 uM (0.000976562μM**)% Activity at given concentration.Float%
18Activity at 0.00214 uM (0.00214233μM**)% Activity at given concentration.Float%
19Activity at 0.00428 uM (0.00427865μM**)% Activity at given concentration.Float%
20Activity at 0.00781 uM (0.0078125μM**)% Activity at given concentration.Float%
21Activity at 0.00940 uM (0.00939941μM**)% Activity at given concentration.Float%
22Activity at 0.018 uM (0.0184361μM**)% Activity at given concentration.Float%
23Activity at 0.034 uM (0.0342772μM**)% Activity at given concentration.Float%
24Activity at 0.062 uM (0.0625μM**)% Activity at given concentration.Float%
25Activity at 0.100 uM (0.100087μM**)% Activity at given concentration.Float%
26Activity at 0.150 uM (0.150391μM**)% Activity at given concentration.Float%
27Activity at 0.274 uM (0.274218μM**)% Activity at given concentration.Float%
28Activity at 0.543 uM (0.543354μM**)% Activity at given concentration.Float%
29Activity at 1.000 uM (1μM**)% Activity at given concentration.Float%
30Activity at 1.203 uM (1.20312μM**)% Activity at given concentration.Float%
31Activity at 2.341 uM (2.34124μM**)% Activity at given concentration.Float%
32Activity at 4.387 uM (4.38748μM**)% Activity at given concentration.Float%
33Activity at 11.33 uM (11.3284μM**)% Activity at given concentration.Float%
34Activity at 19.25 uM (19.25μM**)% Activity at given concentration.Float%
35Activity at 38.50 uM (38.5μM**)% Activity at given concentration.Float%
36Activity at 66.67 uM (66.6667μM**)% Activity at given concentration.Float%
37Activity at 77.00 uM (77μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084827-01

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