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].

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.

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].

Grant Number: 1R03MH084827-01