AID	Name	Type	Probe	Comment
1770	qHTS Assay for Inhibitors of CDC-like Kinase 4 (Kinase-Glo Assay)	confirmatory
488872	qHTS for Inhibitors of DYRK1A: Summary	summary	1
1997	qHTS for Inhibitors of CDC-like Kinase 4: Summary	summary	3
488883	qHTS for Inhibitors of DYRK1B: Summary	summary	1
488887	qHTS for Inhibitors of CDC-like Kinase 1 and 4: Summary	summary	1
493206	Assay for Inhibitors of Dual-Specificity Tyrosine-(Y)-Phosphorylation Regulated Kinase 1A (Kinase-Glo assay): round 2 SAR	confirmatory

NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: 1R03MH084827-01
PI Name: Tom Misteli

NCGC Assay Overview:

The CMGC group of protein kinases which includes CDK-like kinases (Clk) and Dyks are essential kinases found in all eukaryotes. Clks have been implicated to play a role in the regulation of alternative splicing [1]; while Dyrks, in particular over expression of Dyrk1A, has been linked to diseases as Down syndrome (DS; OMIM 190685) [2] and Alzheimer disease (AD; OMIM 104300) [3]. Among the members of the CMGC group, the Clks and the Dyrks are very closely related [4] therefore to determine validity and selectivity of an active compound observed in the CLK4 qHTS screen (AID:1770), a bioluminescent counter-screen against Dyrk1A was developed. In addition, potent and selective compounds against Dyrk1A were identified to further assess the role of this enzyme in DS and AD.
Dyrk1A (Invitrogen, cat # PV3785) was assayed using ATP and DYRKtide (AnaSpec, cat# 62698) as substrates. Promega Kinase-Glo (cat# V6712) technology was used to detect the residual ATP following kinetic reaction. The Kinase-Glo Plus contains Ultra-Glo [5] luciferase and D-luciferin which generates a bioluminescence signal from the remaining ATP. No enzyme and harmine (Sigma, cat # 286044-1G), a compound shown to inhibit Dyrks [6] were used as a positive control; while DMSO treatment was used as a negative control.

NCGC Assay Protocol Summary:

Two uL/well of substrate solution (8uM Dyrktide, 2uM ATP, 25mM Tris-HCl pH 7.5, 10mM MgCl2 , 0.5mM EGTA, 5mM cystein, 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). Compound solution (23 nL) was transferred to the assay plate using Kalypsys pin tool equipped with a 1536-pin tool. One uL/well enzyme solution (5nM Dyrk1A, 25mM Tris-HCl pH7.5, 10mM MgCl2, 0.5mM EGTA, 5mM cystein, 0.01% Triton X-100 final concentration) was then added using FRD yielding a total kinase reaction volume of 3uL/well. After 2 hours of room temperature incubation, 3uL Kinase-Glo detection reagent was added for a final assay volume of 6uL/well. The plates were spun for 30 seconds (1000 rpm) and incubated at ambient temperature for 2 minutes followed by a luminescence read using Perkin Elmer ViewLux plate reader at 3 seconds 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". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
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.

Grant Number: 1R03MH084827-01