Learn More About Transcreener Method Used for This IRAK4 Assay

Discover IRAK4 Modulators with the Transcreener ADP2 Assay

The Transcreener ADP² Kinase Assay directly measures the ADP produced by IRAK4 (also known as interleukin-1 receptor-associated kinase 4). These ADP measurements allow researchers to effectively determine the activity of the IRAK4 enzyme. The assay provides a powerful tool to screen compound libraries for IRAK4 modulators to help find new therapeutics for a variety of different conditions including autoimmune diseases and cancer.

The kit comes complete with all the detection reagents required to measure activity. IRAK4 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

The Role of IRAK4 As Therapeutic Target

As upstream toll-like receptors activate IRAK4, it phosphorylates IRAK1, stimulating downstream substrates by way of its kinase activity. This cascade leads to the activation of MAPKs and NF-κB and the production of pro-inflammatory cytokines. Further research supports IRAK4 as an innate immunity target. In these studies, IRAK knockout mice experienced little or no inflammation when stressed. It has also been found that patients with IRAK4 mutations have issues with pro-inflammatory cytokine and anti-inflammatory cytokine production.

Compounds that regulate IRAK4 provide a potential avenue for small molecule drugs to treat inflammatory and immune disorders. Some diseases associated with IRAK4 include cancer, lupus, inflammatory bowel disease, arthritis, and diabetes.

Learn how Genentech used the Transcreener ADP² Assay to discover IRAK4 inhibitors.

Direct Detection of ADP to Measure IRAK4 Enzymatic Activity

ADP FP Kinase Assay

The workhorse. Used in many large screens. Best deck and signal stability.

ADP FI Kinase Assay

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

ADP TR-FRET ATPase Assay

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of IRAK4
  • Screen Compound Libraries for IRAK4 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

The Transcreener ADP² IRAK4 Assay is in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

Detection of ADP Under IRAK4 Initial Velocity - FP Readout

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 0.1 µg/µL MBP substrate. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The IRAK4 enzyme reaction took place for 60 minutes at room temperature. The enzymatic reaction was then halted using Stop & Detect Buffer B included in the kit and assay plates were read. Non-productive hydrolysis of ATP occurs in many kinase reactions due to many factors including autophosphorylation and water exploiting the ATP binding pocket of the enzyme.

Human IRAK4 Enzyme Titration

IRAK4 Assay Enzyme Titration

Linear Response

IRAK4 Assay Enzyme Titration Linear Response

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 7.5 nM IRAK4, 10 µM ATP, 0.1 µg/µL MBP substrate for an enzyme reaction of 60 minutes at room temperature. IRAK1/4 inhibitor from R&D Systems Cat. # 5665.

Dose-Response - Known IRAK1/4 Inhibitor

IC50 = 0.691 µM

Robust Assay Yields Quality Data

Z’ measurements using optimized IRAK4 reaction conditions indicate a robust assay.  Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Both reactions have 10 µM ATP, 0.1 µg/µL MBP substrate for an enzyme reaction of 60 minutes at room temperature. Z' shown here = 0.77.

IRAK4 Assay Z prime data

IRAK4 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your IRAK4 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
bioteklogo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

TOP