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BioAssay: AID 1983

Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)

Clk4 (Invitrogen cat # PV3839) was assayed using ATP and the RS repeat peptide (AnaSpec cat # 61722) as substrates using Transcreener (trade mark), a competitive fluorescence polarization (FP) assay [1]. For the present assay, we used the Orange TranscreenerTM ADP2 (BellBrooks Labs, Madison, Wis) detection system to monitor ADP production in the kinase reaction. In the assay, an ADP tracer more ..
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(46)
 
 
Inactive(3)
 
 
Inconclusive(14)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(46)
 
 
Inactive(3)
 
 
Inconclusive(14)
 
 
AID: 1983
Data Source: NCGC (CLK268)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-10-14

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: 46
Depositor Specified Assays
AIDNameTypeProbeComment
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)confirmatory
1771qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)confirmatory
1969Confirmaiton Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)confirmatory
1970Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)confirmatory
1379Counterscreen for Luciferase (Kinase-Glo TM) Inhibitionconfirmatory
1795qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)confirmatory
1997qHTS for Inhibitors of CDC-like Kinase 4: Summarysummary3
493204Confirmation Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay): SAR round 2confirmatory
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 using Transcreener (trade mark), a competitive fluorescence polarization (FP) assay [1]. For the present assay, we used the Orange TranscreenerTM ADP2 (BellBrooks Labs, Madison, Wis) detection system to monitor ADP production in the kinase reaction. In the assay, an ADP tracer exhibiting orange fluorescence is bound to an ADP antibody which can be displaced by the ADP product of the kinase reaction. This allows the ADP tracer to freely rotate leading to a decrease in the observed FP value. The amount of ADP produced can then be calculated using a calibration curve. The positive control for the assay was TG003 (Sigma-Aldrich, cat # 300801-52-9) which is a Clk4 inhibitor [2].
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 1,536-well assay plates (Greiner, solid black medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Using a Kalypsys pin tool equipped with a 1,536-pin tool, 23 nL of compound solution was transferred to the assay plate. Two uL/well Clk4-buffer solution (6.25 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 concentration) was then added using the FRD yielding a total kinase reaction volume of 4 uL/ well. Then 4 uL/well of detection mixture (4 nM orange fluorescent tracer, 1 ug/mL ADP antibody, final concentration) was then added after an hour and the polarization signal was measured using the EnVision plate reader (PerkinElmer, Waltham, MA) after 30 min of room temperature incubation using the following filters and program settings: BODIPY TMR FP dual Enh mirror (barcode 682); BODIPY-TMR 531 (barcode 105) excitation; BODIPY-TMR FP P/S polarized 595 nm filters (barcodes 210 and 211, respectively); 100% light; 150 flashes; 1.35 G-factor; Detector gains were 685 for the 1st detector; 630 for the 2nd detector.
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.
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 0.0000001686 uM (1.68588e-07μM**)% Activity at given concentration.Float%
15Activity at 0.0000004768 uM (4.76837e-07μM**)% Activity at given concentration.Float%
16Activity at 0.0000009537 uM (9.53672e-07μM**)% Activity at given concentration.Float%
17Activity at 0.0000019074 uM (1.90735e-06μM**)% Activity at given concentration.Float%
18Activity at 0.0000038147 uM (3.8147e-06μM**)% Activity at given concentration.Float%
19Activity at 0.0000096121 uM (9.61212e-06μM**)% Activity at given concentration.Float%
20Activity at 0.0000193992 uM (1.93992e-05μM**)% Activity at given concentration.Float%
21Activity at 0.0000539987 uM (5.39987e-05μM**)% Activity at given concentration.Float%
22Activity at 0.0001510319 uM (0.000151032μM**)% Activity at given concentration.Float%
23Activity at 0.0003020638 uM (0.000302064μM**)% Activity at given concentration.Float%
24Activity at 0.0006041277 uM (0.000604128μM**)% Activity at given concentration.Float%
25Activity at 0.00123 uM (0.00123035μM**)% Activity at given concentration.Float%
26Activity at 0.00248 uM (0.0024831μM**)% Activity at given concentration.Float%
27Activity at 0.00691 uM (0.00691183μM**)% Activity at given concentration.Float%
28Activity at 0.019 uM (0.0193321μM**)% Activity at given concentration.Float%
29Activity at 0.039 uM (0.0386642μM**)% Activity at given concentration.Float%
30Activity at 0.077 uM (0.0773283μM**)% Activity at given concentration.Float%
31Activity at 0.157 uM (0.157485μM**)% Activity at given concentration.Float%
32Activity at 0.326 uM (0.325581μM**)% Activity at given concentration.Float%
33Activity at 0.892 uM (0.891707μM**)% Activity at given concentration.Float%
34Activity at 2.496 uM (2.4961μM**)% Activity at given concentration.Float%
35Activity at 4.992 uM (4.99221μM**)% Activity at given concentration.Float%
36Activity at 9.984 uM (9.98441μM**)% Activity at given concentration.Float%
37Activity at 19.97 uM (19.9688μM**)% Activity at given concentration.Float%
38Activity at 40.98 uM (40.9792μM**)% Activity at given concentration.Float%
39Activity at 80.00 uM (80μM**)% 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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