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Identification of Kinase-substrate Pairs Using High Throughput Screening

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Cellular Ser/Thr-kinase assays using generic peptide substrates.

Deanna G Adams¹, Yu Wang, Puiying A Mak

¹Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.

Current Chemical Genomics

|February 18, 2010

Summary

This summary is machine-generated.

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A new cellular assay platform enables rapid screening of Serine/Threonine-kinases, crucial for drug discovery. This homogeneous time-resolved fluorescence (TR-FRET) assay quantifies intracellular phosphorylation, accelerating lead identification and kinase inhibitor profiling.

Area of Science:

Biochemistry
Molecular Biology
Drug Discovery

Background:

High-throughput cellular profiling accelerates drug discovery by aiding lead identification and analysis.
Existing universal assay formats effectively target many drug classes but lack a broad solution for Serine/Threonine-kinases.
Rapid and broad interrogation of the Ser/Thr-kinase space remains a challenge in drug discovery pipelines.

Purpose of the Study:

To establish a novel cellular assay platform for broad and rapid interrogation of Serine/Threonine-kinases.
To develop a homogeneous time-resolved fluorescence (TR-FRET) assay for quantifying intracellular substrate phosphorylation.
To validate the platform's utility for kinase profiling and drug discovery.

Main Methods:

Coexpression of target kinases with promiscuous peptide substrates in cellular assays.

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Identification of Kinase-substrate Pairs Using High Throughput Screening

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Quantification of intracellular substrate phosphorylation using homogeneous time-resolved fluorescence (TR-FRET).

Proof-of-concept validation using AKT, B-RAF, and CamK2delta assays, and comparison with gold-standard proliferation assays.

Main Results:

Demonstrated proof-of-concept for cellular AKT, B-RAF, and CamK2delta assays using the TR-FRET platform.
Observed comparable activity profiles for B-Raf inhibitors using peptide or protein substrates.
Showcased strong correlation of IC50 values between cellular TR-FRET and gold-standard proliferation assays for B-Raf.

Conclusions:

The developed cellular Ser/Thr-kinase platform provides a foundation for broad and rapid kinase interrogation.
The TR-FRET assay enables efficient quantification of intracellular phosphorylation, supporting drug discovery efforts.
The platform successfully identified starting points for over 20 additional cellular Ser/Thr-kinase assays.