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BioAssay: AID 1970

Confirmation 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(168)
 
 
Active(157)
 
 
Inactive(1)
 
 
Inconclusive(10)
 
 
 Tested Substances
 Tested Substances
All(169)
 
 
Active(158)
 
 
Inactive(1)
 
 
Inconclusive(10)
 
 
AID: 1970
Data Source: NCGC (CLK807)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-10-09
Modify Date: 2010-10-21

Data Table ( Complete ):           Active    All
Target
Sequence: CDC-like kinase 4 [Homo sapiens]
Description ..   
Protein Family: Protein Kinases, catalytic domain

Gene:CLK4     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 157
Depositor Specified Assays
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AIDNameTypeProbeComment
1379Counterscreen for Luciferase (Kinase-Glo TM) Inhibitionconfirmatory
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)confirmatory
1983Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)confirmatory
1997qHTS for Inhibitors of CDC-like Kinase 4: Summarysummary3
488883qHTS for Inhibitors of DYRK1B: Summarysummary1
504424Kinase Inhibition Study on Inhibitors of Dyrk1a (Reaction Biology data)confirmatory
504427Kinase Inhibition Study on Inhibitors of CDC-like Kinase 2 (Reaction Biology data)confirmatory
504430Kinase Inhibition Study on Inhibitors of CDC-like Kinase 1 (Reaction Biology data)confirmatory
488887qHTS for Inhibitors of CDC-like Kinase 1 and 4: Summarysummary1
493206Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay): round 2 SARconfirmatory
504429Kinase Inhibition Study on Inhibitors of Dyrk1b (Reaction Biology data)confirmatory
488872qHTS for Inhibitors of DYRK1A: Summarysummary1
504421Kinase Inhibition Study on Inhibitors of CDC-like Kinase 4 (Reaction Biology data): SAR round 2confirmatory
2710Kinase Inhibition Profile Study on Inhibitors of CDC-like Kinase 4other
504428Kinase Inhibition Study on Inhibitors of CDC-like Kinase 3 (Reaction Biology data)confirmatory
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].
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 0.0000006743 uM (6.74349e-07μM**)% Activity at given concentration.Float%
15Activity at 0.0000019073 uM (1.90735e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000038147 uM (3.8147e-06μM**)% Activity at given concentration.Float%
17Activity at 0.0000079816 uM (7.98156e-06μM**)% Activity at given concentration.Float%
18Activity at 0.0000159631 uM (1.59631e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0000319262 uM (3.19262e-05μM**)% Activity at given concentration.Float%
20Activity at 0.0000638525 uM (6.38525e-05μM**)% Activity at given concentration.Float%
21Activity at 0.0001310584 uM (0.000131058μM**)% Activity at given concentration.Float%
22Activity at 0.0003604615 uM (0.000360462μM**)% Activity at given concentration.Float%
23Activity at 0.0009591030 uM (0.000959103μM**)% Activity at given concentration.Float%
24Activity at 0.00220 uM (0.00219834μM**)% Activity at given concentration.Float%
25Activity at 0.00409 uM (0.00408656μM**)% Activity at given concentration.Float%
26Activity at 0.00806 uM (0.00806401μM**)% Activity at given concentration.Float%
27Activity at 0.018 uM (0.0182213μM**)% Activity at given concentration.Float%
28Activity at 0.033 uM (0.0326925μM**)% Activity at given concentration.Float%
29Activity at 0.087 uM (0.087278μM**)% Activity at given concentration.Float%
30Activity at 0.240 uM (0.240285μM**)% Activity at given concentration.Float%
31Activity at 0.545 uM (0.544521μM**)% Activity at given concentration.Float%
32Activity at 1.046 uM (1.04616μM**)% Activity at given concentration.Float%
33Activity at 2.032 uM (2.03226μM**)% Activity at given concentration.Float%
34Activity at 4.592 uM (4.59206μM**)% Activity at given concentration.Float%
35Activity at 8.386 uM (8.38571μM**)% Activity at given concentration.Float%
36Activity at 16.87 uM (16.8708μM**)% Activity at given concentration.Float%
37Activity at 47.71 uM (47.7127μM**)% Activity at given concentration.Float%
38Activity at 77.00 uM (77μM**)% Activity at given concentration.Float%
39Activity at 153.8 uM (153.846μM**)% 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: 1R03MH084827-01

Data Table (Concise)
Classification
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