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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

ATLAS--a high-throughput affinity-based screening technology for soluble proteins: technology application using p38

Rupal Patel¹, Laurie A Lebrun, Shaohui Wang

¹Department of Biology, Anadys Pharmaceuticals, Inc., San Diego, California, USA.

Assay and Drug Development Technologies

|March 14, 2008

Summary

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A novel Any Target Ligand Affinity Screen (ATLAS) assay identifies drug compounds by detecting protein stabilization against thermal unfolding. This high-throughput method aids in discovering lead compounds for various protein targets, including difficult-to-assay ones.

Area of Science:

Biochemistry
Drug Discovery
Molecular Biology

Background:

Protein thermal unfolding and aggregation are key indicators of molecular instability.
Identifying compounds that bind and stabilize target proteins is crucial for drug discovery.
Existing screening methods may not be suitable for all protein targets, especially those difficult to assay functionally.

Purpose of the Study:

To describe a general affinity-based screening assay for discovering lead compounds targeting proteins.
To introduce the Any Target Ligand Affinity Screen (ATLAS) technology for high-throughput screening.
To demonstrate the applicability of ATLAS for proteins that are difficult to assay functionally.

Main Methods:

The ATLAS assay utilizes protein thermal unfolding and aggregation as a basis for screening.

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It employs time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-(His)6 antibodies to detect aggregated hexahistidine-tagged proteins.

The assay is homogeneous, simple, and automatable for screening large compound libraries.

Main Results:

ATLAS successfully identified compounds that bind and protect target proteins from thermal unfolding and aggregation.
The assay demonstrated good correlation with a functional assay when tested with known inhibitors of p38 mitogen-activated protein (MAP) kinase.
The technology is applicable to soluble proteins of known and unknown functions.

Conclusions:

ATLAS is a versatile and efficient technology for lead discovery of protein-binding compounds.
It offers a robust method for screening targets that are challenging for traditional functional assays.
The correlation with functional assays validates ATLAS as a reliable screening tool in drug discovery.