Dyrk1 A HTS Measured in Biochemical System Using Plate Reader - 2124-01_Inhibitor_SinglePoint_HTS_Activity
Assay Overview: The goal of this project is to develop small molecule modulators of the Nuclear Factor Activated T-cells (NFAT) signaling by identifying inhibitors of Dyrk1A kinase. Using the Promega ADP-Glo Kinase Assay kit, a low volume, 1536-well assay will be used to screen the MLSMR. Small-molecule inhibitors of Dyrk1A kinase activity will be identified and tested in the assay critical more ..
BioActive Compounds: 1321
Keywords: Dyrk-1 kinase, ADP-Glo Kinase Assay, Nuclear Factor Activated T-cells, NFAT
Assay Overview: The goal of this project is to develop small molecule modulators of the Nuclear Factor Activated T-cells (NFAT) signaling by identifying inhibitors of Dyrk1A kinase. Using the Promega ADP-Glo Kinase Assay kit, a low volume, 1536-well assay will be used to screen the MLSMR. Small-molecule inhibitors of Dyrk1A kinase activity will be identified and tested in the assay critical path. Dyrk1 A kinase inhibitors that do not inhibit the luciferase component of the original screening assay and demonstrate an IC50 < 10 uM against Dyrk1 A kinase will be evaluated for selectivity against CLK1 and ERK kinases. The resulting potent, selective inhibitors of Dyrk1 A kinase could lead to the development of novel probes for chemical genetic studies of development. They could also become potential drug leads for a variety of clinically relevant conditions, most notably Down Syndrome.
Expected Outcome: In this assay, Dyrk1 A kinase transfers a phosphate group from ATP to a peptide substrate, NFAT C1. Inhibitors of Dyrk1 A kinase will mimic the no enzyme control and result in loss of luminescence signal in the Promega ADP-Glotrade mark Kinase Assay. This procedure is a luminescent ADP detection assay that provides a homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction. The assay is performed in two steps; first, after the kinase reaction, an equal volume of ADP-Glotrade mark Reagent is added to terminate the kinase reaction and deplete the remaining ATP. Second, the Kinase Detection Reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The light generated is measured using a luminometer. A kinase inhibitor will demonstrate a decrease in luminescence relative to an uninhibited control.
1.) 1536-well assay ready white plates (Aurora 00019210) with a 2.5 nL compound spot were used as the reaction plates. The 2.5 nL spot results in a 12.5 uM final compound concentration and 0.125% DMSO in the reaction.
2.) 2 solutions were mixed to initiate the reaction. The substrate solution contained 20 uM ATP and 22 uM NFAT C1 peptide ([H]-GAR-SSR-PAS-PCN-KRK-[OH], Tufts Peptide Synthesis) in a reaction buffer containing: 20 mM HEPES, 10 mM MgCl2, 50 uG/ml BSA, 1 mM DTT, pH 7.5. The enzyme solution contained Dyrk1 A kinase diluted to 2 ng in 20 mM HEPES, 10 mM MgCl2, pH 7.5. 1 uL of each solution was added simultaneously by the BioRaptor liquid handler to the ARP reaction plate and allowed to incubate at 28 degrees C for 2 hrs. The final reactant concentrations were 1 ng Dyrk1 A, 10 uM ATP, 11uM NFAT C1 peptide.
3.) Control wells contained no compound reactions with 0.125% DMSO, 1 ng Dyrk1A (final) or no enzyme buffer.
4.) After the 2 hr. incubation, the reaction was terminated by the addition of 2 uL ADP-Glo Reagent (Promega, #V9102). This reaction also depletes the remaining ATP. The stopped reaction is allowed to incubate for 40 mins.
5.) After 40 mins., 4 uL of Kinase Detection Reagent ( Promega, #V9102) is added to simultaneously convert ADP to ATP and allow newly synthesized ATP to be measured using a luciferase/luciferin reaction.
6.) The reaction was allowed to proceed for 30 mins. and luminescence was detected using a Perkin Elmer View Lux (model 1430-002).
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
This was set as equal to the maximum of the normalized sample activity, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 50.
Samples passing threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT
Samples passing AT were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)