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Updated: Jul 19, 2025

Identification of Kinase-substrate Pairs Using High Throughput Screening

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

A biophysical framework for double-drugging kinases.

Chansik Kim^1,2, Hannes Ludewig^1,2, Adelajda Hadzipasic^1,2

¹Department of Biochemistry, Brandeis University, Waltham, MA 02454.

Proceedings of the National Academy of Sciences of the United States of America

|August 17, 2023

Summary

This summary is machine-generated.

Double-drugging kinases by simultaneously targeting orthosteric and allosteric sites offers a synergistic approach to overcome drug resistance. This strategy enhances drug efficacy and reduces required dosages for Aurora A kinase and Abelson kinase inhibition.

Keywords:

conformational equilibrium cooperativity double-drugging kinase

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Last Updated: Jul 19, 2025

Identification of Kinase-substrate Pairs Using High Throughput Screening

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Published on: August 29, 2015

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

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Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Published on: October 23, 2019

Area of Science:

Biochemistry
Structural Biology
Pharmacology

Background:

Selective kinase inhibition is difficult due to conserved active sites and resistance mutations.
Simultaneous targeting of orthosteric and allosteric sites ('double-drugging') shows promise for overcoming resistance.
The cooperative mechanisms underlying kinase double-drugging require detailed biophysical characterization.

Purpose of the Study:

To develop a quantitative framework for characterizing kinase double-drugging.
To investigate the cooperative effects between orthosteric and allosteric modulators on Aurora A kinase (AurA) and Abelson kinase (Abl).
To elucidate the structural and mechanistic basis of synergistic kinase inhibition.

Main Methods:

Isothermal titration calorimetry (ITC)
Förster resonance energy transfer (FRET)
Coupled-enzyme assays
X-ray crystallography

Main Results:

Identified positive and negative cooperativity between orthosteric and allosteric modulators for AurA and Abl.
Demonstrated that conformational equilibrium shifts govern cooperativity.
Observed synergistic reduction in drug dosages for clinically relevant inhibition levels.
Determined X-ray crystal structures of double-drugged kinase complexes, revealing molecular mechanisms.
Reported a fully closed Abl conformation induced by positively cooperative modulators.

Conclusions:

Double-drugging provides a synergistic strategy for kinase inhibition, reducing drug dosage requirements.
Conformational equilibrium shifts are key to understanding cooperativity in double-drugging.
Mechanistic and structural insights support the rational design of double-drugging therapies.