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Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery
Yiping Chen1, Dongyu Sun1, Ruojing Yang1
1Department of Quantitative Bioscience, Merck & Co., Inc., Boston, MA, USA.
Abstract:
One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.
Insights
Developing a novel assay for Interleukin-1 receptor-associated kinase 4 (IRAK4) engagement improves preclinical drug discovery. This proximal cellular assay aids in identifying selective IRAK4 inhibitors for inflammatory diseases, enhancing therapeutic success rates.
Area of Science:
- Pharmacology and Drug Discovery
- Immunology and Inflammation
Background:
- Lack of specific target engagement is a major cause of drug failure in late-stage clinical trials.
- Early preclinical assessment of proximal target engagement can increase the success rate of new therapeutics.
- Interleukin-1 receptor-associated kinase 4 (IRAK4) is a kinase implicated in inflammatory and autoimmune diseases.
Purpose of the Study:
- To establish a proximal, functional assay for evaluating IRAK4 target engagement in a cellular context.
- To support structure-activity relationship (SAR) studies for the identification and optimization of selective IRAK4 inhibitors.
- To validate the biological relevance of IRAK1 activation as a biomarker for IRAK4 activity.
Main Methods:
- Development of an electrochemiluminescence (ECL)-based cellular assay measuring endogenous IRAK1 activation.
- Utilized IRAK1 activation assay to assess IRAK4 engagement across different compound classes.
- Correlated assay data with IRAK4 biochemical kinase activity and TNFα secretion assays from peripheral blood mononuclear cells (PBMCs).
Main Results:
- The developed IRAK1 activation assay demonstrated statistically significant correlations with IRAK4 kinase activity and functional PBMC assays.
- Assay validation confirmed IRAK1 engagement as a relevant biomarker for IRAK4 activity.
- The assay exhibited plate uniformity and potency reproducibility, indicating suitability for high-throughput screening.
Conclusions:
- The proximal cellular IRAK1 activation assay is a valuable tool for early preclinical assessment of IRAK4 inhibitors.
- Incorporating this assay into in vitro screening can prevent optimization towards off-target activities.
- This approach enhances the likelihood of developing successful therapeutics for inflammatory and autoimmune diseases.
