AID	Name	Type	Probe	Comment
1379	Counterscreen for Luciferase (Kinase-Glo TM) Inhibition	confirmatory
1771	qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-Glo Assay)	confirmatory
1770	qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)	confirmatory
1795	qHTS Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)	confirmatory
1983	Confirmation Assay for Inhibitors of CDC-like Kinase 4 (ADP-FP Assay)	confirmatory
1997	qHTS for Inhibitors of CDC-like Kinase 4: Summary	summary	3

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].

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.

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