Genentech discovers IRAK4 inhibitors using the Transcreener Kinase Assay Kit – IRAK is also known as Interluekin-1 Receptor Associated Kinase. There are four different proteins within this group, including IRAK1, IRAK2, IRAK3, and IRAK4. Each of these mediates interaction with MyD88 and its associated adaptor proteins. IRAK1 and IRAK4 are active enzymes that exhibit kinase activity, while IRAK3 is generally considered inactive. IRAK2 was once thought to be inactive, but research has shown its kinase activity to be critical for TLR signaling.3
As upstream toll-like receptors activate IRAK4, it phosphorylates IRAK1. IRAK1 then autophosphorylates and activates 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.2 Further research supports IRAK4 as an innate immunity target. In these studies, IRKA1 knockout mice experienced little or no inflammation when subjected to “experimental autoimmune encephalomyelitis.”3 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 to small molecule drugs to treat inflammatory and immune disorders. Some diseases associated with IRAK4 include cancer, lupus, inflammatory bowel disease, arthritis, and diabetes.1
Using A Kinase Assay Kit in Drug Discovery
In the recently issued patent, Genentech discusses how it used the Transcreener Kinase Assay Kit to measure enzymatic activity and determine Ki values for various compounds. In summary, IRAK4 assay conditions were tested with a synthesized IRAK1 peptide used as a substrate. Samples were incubated 90 minutes, and a target 10% conversion of ATP to ADP was achieved. The sensitivity of the assay allowed researchers to use low nM concentrations of IRAK4 (1.25 nM). IRAK1 activity was also measured using the same synthesized peptide substrate previously discussed. 10% conversion was also used with starting ATP concentrations of 62.5 uM. Assays were then read on Tecan M1000 plate readers, and data were subsequently analyzed. Examples of nanomolar inhibitors are illustrated.
Using kinase assay kits to measure enzymatic activity is critical to finding novel inhibitors during a high throughput screen. Assays must be robust enough to be handled for hours during large screens but accurate enough to provide solid, reproducible data with good sensitivity. As illustrated by Genentech, follow-up SAR is essential to building the right set of compounds that lead to therapeutics in the clinic. A biochemical kinase assay kit like Transcreener is a critical component when discovery and solidifying novel inhibitors into leads. IRAK4 is a compelling innate immune target, and it will be interesting to see how clinical trials uncover potential therapeutics for diseases such as Rheumatoid Arthritis.
(1) Bryan, Marian C. Drobnick, Joy Gobbi, Alberto Katsumoto, Tamiko Rajapaksa, Naomi S. Kiefer Jr., James Richard. 2020 IRAK4 MODULATORS Genentech, Inc. (South San Francisco, CA, US) United States Patent.: 20200115388 https://www.freepatentsonline.com/y2020/0115388.html
(2) Li, Shyun, Astrid Strelow, Elizabeth J. Fontana, and Holger Wesche. “IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase.” Proceedings of the National Academy of Sciences 99, no. 8 (2002): 5567-5572. https://www.pnas.org/content/99/8/5567.short
(3) Flannery, Sinead, and Andrew G. Bowie. “The interleukin-1 receptor-associated kinases: critical regulators of innate immune signalling.” Biochemical pharmacology 80, no. 12 (2010): 1981-1991. https://www.sciencedirect.com/science/article/abs/pii/S0006295210004545