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Updated: Aug 14, 2026
Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Published on: October 23, 2019
Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification
Yong Jia1, Christopher M Quinn, Anca Clabbers
1Department of Molecular Pharmacology, Abbott Bioresearch Center, Worcester, MA 01605, USA. yong.jia@abbott.com
Abstract:
Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.
Insights
Cancer Osaka Thyroid (COT) kinase assays were developed for anti-inflammatory drug discovery. Homogeneous Time-Resolved Fluorescence (HTRF) assays using peptide substrates are preferred for robust screening and mechanistic studies.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Drug discovery
Background:
- Cancer Osaka Thyroid (COT) is a kinase involved in tumor necrosis factor-alpha production.
- COT is a potential target for anti-inflammatory drug development.
- Development of reliable in vitro assays is crucial for identifying COT inhibitors.
Purpose of the Study:
- To develop and compare different in vitro assay formats for Cancer Osaka Thyroid (COT).
- To identify optimal assay conditions for compound screening and mechanistic studies.
- To establish methods for assessing inhibitor ATP competitiveness.
Main Methods:
- Development of cascade and direct in vitro assays for COT.
- Comparison of radiometric and homogeneous time-resolved fluorescence (HTRF) assay formats.
- Utilized MEK1 protein and peptide substrates to evaluate inhibitor potency.
- Developed a method to assess ATP competitiveness of inhibitors.
Main Results:
- Cascade assays are suitable for high-throughput screening but not mechanistic studies.
- Direct assays, particularly HTRF, are superior for both screening and mechanistic studies.
- Using MEK1 peptide substrates eliminated false positives observed with MEK1 protein substrate.
- A method for assessing ATP competitiveness of inhibitors was successfully developed.
Conclusions:
- HTRF assays using MEK1 peptide substrates provide a robust platform for COT inhibitor screening and characterization.
- The developed methods are applicable to other kinase targets in drug discovery.
- Accurate assessment of inhibitor properties requires careful substrate selection and mechanistic studies.
