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BioAssay: AID 1969

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

Clk4 (Invitrogen, cat# PV3839) was assayed using ATP and the RS repeat peptide (AnaSpec cat # 61722) as substrates. In the assay, the ADP levels were detected using Promega ADP-Glo technology wherein the remaining ATP from the kinase reaction is first depleted with an ATPase reagent followed by a reagent that contains and enzyme which converts the ADP produced to ATP as well as Ultra-Glo more ..
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 Tested Compounds
 Tested Compounds
All(63)
 
 
Active(57)
 
 
Inactive(1)
 
 
Inconclusive(5)
 
 
 Tested Substances
 Tested Substances
All(63)
 
 
Active(57)
 
 
Inactive(1)
 
 
Inconclusive(5)
 
 
AID: 1969
Data Source: NCGC (CLK946)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-10-09

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: 57
Depositor Specified Assays
AIDNameTypeProbeComment
1379Counterscreen for Luciferase (Kinase-Glo TM) Inhibitionconfirmatory
1771qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)confirmatory
1770qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)confirmatory
1795qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)confirmatory
1983Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)confirmatory
1997qHTS for Inhibitors of CDC-like Kinase 4: Summarysummary3
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. In the assay, the ADP levels were detected using Promega ADP-Glo technology wherein the remaining ATP from the kinase reaction is first depleted with an ATPase reagent followed by a reagent that contains and enzyme which converts the ADP produced to ATP as well as 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, 1x ADP-Glo Buffer A, 2 mM MgCl2, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100, final concentrations) was dispensed into 1536-well plates (Greiner, solid white, medium binding assay plates) with the Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Twenty-three nanoliter of compound and control solutions were transferred to the assay plate using a Kalypsys pin-tool followed by 0.5 uL/well Clk4-buffer solution (25 nM Clk4, 1x ADP-Glo Buffer A, 2 mM MgCl2, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100 final concentration) dispensing for a total kinase reaction volume of 2.5 uL/ well. After 1 hr of room-temperature incubation, 2.5 uL ADP-Glo reagent was added and incubated at room temperature for 45 min to stop the kinetic reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 5 uL per well of ADP-Glo Reagent II to yield a total assay volume of 10 uL/ well. Luminescence was detected after 30 min room temperature incubation with the Perkin Elmer Viewlux.
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 0.0000019074 uM (1.90735e-06μM**)% Activity at given concentration.Float%
16Activity at 0.0000038147 uM (3.8147e-06μM**)% Activity at given concentration.Float%
17Activity at 0.0000094335 uM (9.43345e-06μM**)% Activity at given concentration.Float%
18Activity at 0.0000188669 uM (1.88669e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0000377338 uM (3.77338e-05μM**)% Activity at given concentration.Float%
20Activity at 0.0000754676 uM (7.54676e-05μM**)% Activity at given concentration.Float%
21Activity at 0.0001509352 uM (0.000150935μM**)% Activity at given concentration.Float%
22Activity at 0.0003018704 uM (0.00030187μM**)% Activity at given concentration.Float%
23Activity at 0.0006037408 uM (0.000603741μM**)% Activity at given concentration.Float%
24Activity at 0.00123 uM (0.00123093μM**)% Activity at given concentration.Float%
25Activity at 0.00249 uM (0.00248573μM**)% Activity at given concentration.Float%
26Activity at 0.00497 uM (0.00497145μM**)% Activity at given concentration.Float%
27Activity at 0.010 uM (0.0101995μM**)% Activity at given concentration.Float%
28Activity at 0.028 uM (0.0278818μM**)% Activity at given concentration.Float%
29Activity at 0.077 uM (0.0772788μM**)% Activity at given concentration.Float%
30Activity at 0.155 uM (0.154558μM**)% Activity at given concentration.Float%
31Activity at 0.309 uM (0.309115μM**)% Activity at given concentration.Float%
32Activity at 0.618 uM (0.618231μM**)% Activity at given concentration.Float%
33Activity at 1.236 uM (1.23646μM**)% Activity at given concentration.Float%
34Activity at 2.473 uM (2.47292μM**)% Activity at given concentration.Float%
35Activity at 4.946 uM (4.94584μM**)% Activity at given concentration.Float%
36Activity at 9.983 uM (9.9834μM**)% Activity at given concentration.Float%
37Activity at 19.97 uM (19.9668μM**)% Activity at given concentration.Float%
38Activity at 41.04 uM (41.0412μM**)% 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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