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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
A high throughput assay to identify substrate-selective inhibitors of the ERK protein kinases
Chad J Miller1, Yagmur Muftuoglu1, Benjamin E Turk1
1Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, United States.
Abstract:
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylate a variety of substrates important for survival and proliferation, and their activity is frequently deregulated in tumors. ERK pathway inhibitors have shown clinical efficacy as anti-cancer drugs, but most patients eventually relapse due to reactivation of the pathway. One factor limiting the efficacy of current therapeutics is the difficulty in reaching clinically effective inhibition of the ERK pathway in the absence of on-target toxicities. Here, we describe an assay suitable for high throughput screening to discover substrate selective ERK1/2 inhibitors, which may have a larger therapeutic window than conventional inhibitors. Specifically, we aim to target a substrate-binding pocket within the ERK1/2 catalytic domain outside of the catalytic cleft. The assay uses an AlphaScreen format to detect phosphorylation of a high-efficiency substrate harboring an essential docking site motif. Pilot screening established that the assay is suitably robust for high-throughput screening. Importantly, the assay can be conducted at high ATP concentrations, which we show reduces the discovery of conventional ATP-competitive inhibitors. These studies provide the basis for high-throughput screens to discover new classes of non-conventional ERK1/2 inhibitors.
Insights
We developed a new assay to find selective inhibitors of extracellular signal-regulated kinases 1 and 2 (ERK1/2). This approach aims to overcome drug resistance and reduce toxicity in cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are crucial for cell survival and proliferation.
- Dysregulated ERK pathway activity is common in various cancers, leading to tumor growth.
- Current ERK pathway inhibitors face challenges like drug resistance and on-target toxicities, limiting their clinical efficacy.
Purpose of the Study:
- To develop a high-throughput screening assay for discovering substrate-selective ERK1/2 inhibitors.
- To identify inhibitors targeting a novel substrate-binding pocket outside the catalytic cleft.
- To enable the discovery of non-conventional ERK1/2 inhibitors with a potentially wider therapeutic window.
Main Methods:
- Utilized an AlphaScreen format to detect the phosphorylation of a high-efficiency substrate.
- Designed the substrate to include an essential docking site motif for specific recognition.
- Conducted pilot screening to validate assay robustness for high-throughput screening (HTS).
Main Results:
- The developed assay is robust and suitable for HTS.
- The assay can be performed at high ATP concentrations, which minimizes the identification of ATP-competitive inhibitors.
- Demonstrated the feasibility of targeting substrate-binding pockets distinct from the catalytic cleft.
Conclusions:
- The study presents a novel HTS assay for identifying substrate-selective ERK1/2 inhibitors.
- This approach offers a strategy to discover non-conventional inhibitors that may overcome resistance and reduce toxicity.
- Provides a foundation for future drug discovery efforts targeting the ERK pathway in cancer.
