Assay Method Information
Assay Name:  IRAK4 Biochemical HTRF Kinase Assay
Description:  The binding capacities of certain bifunctional compounds of Formula (I) were determined using the CisBio HTRF KinEASE STK S1 Kit (#62ST1PEB, contains 5× Kinase buffer, 1× Detection Buffer, anti-phospho-serine/threonine-cryptate, Streptavidin-XL665, and STK-S1), which measures the phosphorylation of a biotinylated peptide substrate by IRAK4, according to the manufacturer's protocol. Briefly, test compounds in DMSO were serially diluted into 384 Plus White Proxiplate (PerkinElmer, #6008280) using a Labcyte Echo 550 Liquid Handler, at a concentration of 50× final in 200 nL of 100% DMSO. 7.8 μl of kinase solution, containing 1.28 nM IRAK4 in 1× Kinase Buffer (supplemented with 3 mM MgCl2, 0.01% Triton X-100, and 1 mM DTT) was added to each compound containing well and incubated at ambient temperature for 30 min. 2 μl of a reaction solution containing 5 μM STK-S1, 10 mM ATP, and 10 mM MgCl2 were added to each well to a final volume of 10 μl. Assay controls include wells containing kinase with no compound (DMSO only) and wells containing no kinase and no compound (DMSO only). The reactions were allowed to proceed at ambient temperature for 90 minutes. The reactions were stopped by addition of 10 ul Detection Buffer containing 2× anti-phospho-serine/threonine antibody cryptate and 125 nM Streptavidin-XL665. The plates were incubated for 60 minutes at ambient temperature and then read on an Envision Multilabel Reader (PerkinElmer). HTRF ratio was calculated as (acceptor signal at 665 nm/donor signal at 620 nm)×104 and data was normalized to % inhibition using control wells with no compound as 0% and wells with no kinase and no compound as −100% inhibition. For IC50 determination, the compounds were tested at sixteen concentrations in duplicate and curve-fitting was performed by non-linear regression analysis using GraphPad Priam.
Affinity data for this assay
 
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