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 . 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 ..
BioActive Compounds: 46
Depositor Specified Assays
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 . 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 .
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)