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Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay
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Quantitative Irreversible Tethering (qIT) for Target-directed Covalent Fragment Screening.

Gregory B Craven1,2, Alan Armstrong2, David J Mann1

  • 1Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.

Bio-Protocol
|March 4, 2021
PubMed
Summary

This study introduces quantitative irreversible tethering to screen cysteine-reactive fragments for covalent probe development. This method accurately identifies and ranks fragments based on their selective reaction kinetics with target proteins.

Keywords:
Covalent fragmentsCysteine targetingFragment-based drug discoveryIrreversible inhibitionQuantitative irreversible tethering

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Drug Discovery

Background:

  • Covalent small molecules are vital as biological tools and therapeutics.
  • Identifying covalent binders often involves screening cysteine-reactive fragments against protein targets.
  • Mass spectrometry aids in determining covalent binding sites but lacks kinetic characterization.

Purpose of the Study:

  • To develop a robust screening platform for identifying covalent fragment binders.
  • To characterize the kinetics of covalent fragment-protein interactions.
  • To enable efficient development of covalent probes and therapeutic agents.

Main Methods:

  • Utilized a quantitative irreversible tethering platform employing a cysteine-reactive fluorogenic probe.
  • Simultaneously screened fragment libraries against a target protein and glutathione as a control.
  • Monitored the reaction rates between fragments and cysteine-containing biomolecules.

Main Results:

  • The screening platform accurately identifies hit fragments with kinetic selectivity for target protein modification.
  • Quantitative irreversible tethering accounts for intrinsic fragment reactivity, enabling robust hit ranking.
  • This method facilitates the identification of starting points for covalent probe development.

Conclusions:

  • Quantitative irreversible tethering is an effective method for screening covalent fragments.
  • The platform provides robust hit identification and ranking by considering binding kinetics.
  • This approach advances the development of targeted covalent inhibitors and chemical probes.